(Received for publication, November 20, 1995)
From the
Calcium release from intracellular stores occurs in a graded
manner in response to increasing concentrations of either inositol
1,4,5-trisphosphate or caffeine. To investigate the mechanism
responsible for this quantal release phenomenon,
[Ca] changes inside intracellular stores in
isolated single smooth muscle cells were monitored with mag-fura 2.
Following permeabilization with saponin or
-toxin the dye, loaded
via its acetoxymethyl ester, was predominantly trapped in the
sarcoplasmic reticulum (SR). Low caffeine concentrations in the absence
of ATP induced only partial Ca
release; however,
after inhibiting the calcium pump with thapsigargin the same stimulus
released twice as much Ca
. When the SR
Ca
-ATPase was rendered non-functional by depleting
its ``ATP pool,'' submaximal caffeine doses almost fully
emptied the stores of Ca
. We conclude that quantal
release of Ca
in response to caffeine in these smooth
muscle cells is largely due to the activity of the SR
Ca
-ATPase, which appears to return a portion of the
released Ca
back to the SR, even in the absence of
ATP. Apparently the SR Ca
-ATPase is fueled by ATP,
which is either compartmentalized or bound to the SR.
Cells respond to increasing concentrations of many hormones and
neurotransmitters with graded changes in cytosolic
[Ca]. The rise in
[Ca
] arises in many cells due to release of
Ca
from internal stores through activation of the
inositol 1,4,5-trisphosphate (IP
) (
)receptor by
IP
(1) . The graded nature of Ca
release from internal stores is known as quantal
release(2) . Internal Ca
stores in many cells
also contain ryanodine receptors (RyR), which are activated by
cytoplasmic calcium (3) and possibly cyclic
ADP-ribose(4) . Calcium release due to activation of this
receptor with caffeine also occurs in a quantal
fashion(5, 6) . Three principal hypotheses have been
proposed to explain the partial emptying of Ca
from
internal stores by submaximal levels of IP
or caffeine. 1)
Individual store elements have different sensitivities to agonists and
empty in an all or none fashion (7) ; 2) all stores partially
empty at certain agonist concentrations due to diminished positive
feedback effects of luminal [Ca
] on
Ca
release(7) ; and 3) individual store
elements differ not in the sensitivity of their IP
or
possibly ryanodine receptors but rather in the density of these
receptors(8) .
An alternative to these explanations is that
Ca release is graded in response to increasing levels
of agonists simply because at different levels of stimulation a new
steady state is achieved reflecting IP
- or caffeine-induced
stimulation of release and Ca
sequestration by pumps
associated with these stores. This explanation has been dismissed (9, 10) as IP
-induced Ca
release in permeabilized cells still occurred in a ``quantal
fashion'' under conditions where Ca
pumps were
nominally blocked due to ATP removal. However, evidence is emerging
that the ATP consumed by some ion pumps (11, 12) is
compartmentalized and might not be readily lost either by washing away
ATP or even by incubation with ATP scavengers used in some
studies(9, 10) . If Ca
pumps on
internal stores also have a compartment of inaccessible ATP then the
role of such pumps in quantal release phenomena may have to be
reconsidered. Recent work showing that inhibition of the
Ca
-ATPase with thapsigargin abolishes
IP
-induced quantal release in permeabilized pancreatic
acinar cells (13) suggests that activity of Ca
pumps may well be required for quantal Ca
release. As thapsigargin caused a significant Ca
leak from intracellular stores in that study, the interpretation
of those results may be difficult. Furthermore this study was carried
out on populations of cells, and hence graded release may have arisen
not because release of Ca
in each cell was graded but
because of variations in the responses of cells within the population.
We thus developed a technique to monitor
[Ca
] changes in the sarcoplasmic reticulum
in single isolated smooth muscle cells by loading these cells with the
low affinity calcium indicator mag-fura 2 AM and permeabilizing the
cell membrane to remove cytosolic dye and gain access to the cytoplasm,
a general strategy first described by Hofer and Machen(14) .
In permeabilized cells, mag-fura 2 was distributed in a punctuate manner principally beneath the cell membrane and around the nucleus and was almost absent from the cytoplasm (Fig. 1). The pattern of dye distribution beneath the cell membrane was similar to that of calsequestrin(18) , a calcium-binding protein present in the SR of these cells, indicating that the loaded intracellular stores have a distribution pattern similar to this cellular compartment.
Figure 1:
Three-dimensional distribution of
mag-fura 2-loaded stores in -toxin-permeabilized smooth muscle
cell. Image to the left is a projection of 33 image planes at
0.5-µm intervals through cell; a cross-section is given on the right. Scale bars are 10 µm. Pixel spacing in x and y (0.25 pixel/µm) was less than in z (0.50 pixel/µm) and accounts largely for apparent asymmetry of
the cell cross-section. Larger bright regions are most likely
dye droplets adsorbed to the cell surface. The large oval at
the center of the X-Y projection is dye associated with nuclear
envelope.
Caffeine (25 mM, no ATP), which activates the
RyR in these cells(20) , decreased the fluorescence ratio of
mag-fura 2, and thus the [Ca], in the
intracellular stores from 2.42 to 0.58. These stores apparently
released their total free calcium in response to this high caffeine
concentration as the final fluorescence ratio was virtually identical
to R
in vitro. In the presence of ATP these
stores were capable of calcium uptake, achieving a fluorescence ratio
at least 80% of that prior to the initial caffeine challenge. As can be
seen in Fig. 2A the response to caffeine was quite
reproducible. Refilling of internal stores following caffeine
stimulation was almost completely blocked by the specific inhibitor of
the SR Ca
-ATPase thapsigargin (5 min, 2
µM; Fig. 2B). Thus, based on the
observations that the loaded intracellular stores have an anatomical
distribution similar to the SR, contain a functional RyR and
Ca
-ATPase, and seem to release their total free
calcium content in response to caffeine, we believe that the majority
of mag-fura 2 signal originates from a SR-like compartment.
Figure 2:
Changes in mag-fura 2 fluorescence ratio
in response to caffeine in single saponin-permeabilized smooth muscle
cells. A, two consecutive responses to caffeine in a single
smooth muscle cell. After perfusing chamber with solution A (ATP free)
for 60 s, solution A containing 25 mM caffeine was applied to
the cell, the chamber again perfused with solution A for 60 s, and the
cells placed in solution B (containing ATP) for 5 min. Solution A was
then applied and the cell stimulated again with caffeine as before.
Mag-fura 2 fluorescence decreased in a single exponential manner upon
the first (2.15*(exp.(-0.1917*t)) + 0.2824; r = 0.98) and second
(1.473*(exp.(-0.2297*t)) + 0.3932; r
= 0.99) caffeine (25 mM) stimulation. B,
effect of thapsigargin on the refilling of Ca
stores
as measured with mag-fura 2 in a single cell. Following initial
caffeine stimulation and store refilling, the cell was exposed to
2*10
M thapsigargin for 5 min and then
exposed to caffeine followed by solution B. C, two consecutive
responses to caffeine at a lower concentration (5 mM) in
another smooth muscle cell. Experimental protocol are identical to that
in A.
While
high caffeine concentration (25 mM) caused the stores to
release virtually all stored Ca, lower caffeine
concentrations (5 mM) released less Ca
in a
reproducible manner (Fig. 2C). Increasing the caffeine
concentration from 5 to 25 mM in the absence of ATP caused
additional Ca
release in the same cell (Fig. 3). The resting fluorescence ratio before caffeine
application averaged 1.86 ± 0.13 and decreased to 1.18 ±
0.13 and 0.63 ± 0.05 in the presence of 5 and 25 mM caffeine, respectively (n = 7; Fig. 3B). This dose-dependent graded release of
Ca
from stores in the absence of ATP is a fundamental
characteristic of the ``quantal response'' (12) (Fig. 3A).
Figure 3: Effect of incremental caffeine stimulation on mag-fura 2 fluorescence ratio in single saponin-permeabilized smooth muscle cells. A, typical fluorescence ratio recorded from a cell stimulated with 5 and then 25 mM caffeine. B, mean and S.E. for the steady states in fluorescence ratio in 7 cells subject to protocol in A. *, p < 0.05 compared with control;**, p < 0.05 compared with 5 mM caffeine.
What is responsible for the
graded nature of Ca release with these submaximal
doses of caffeine? It has been suggested that partial emptying of
stores under such conditions may result from the presence of receptors
on individual stores with different sensitivities to caffeine or
alternatively that individual stores may have different densities of
receptors with equivalent sensitivities to caffeine. In either case
release of Ca
in response to supramaximal doses of
caffeine should be described by a multiexponential process. We found,
however, that a single-exponential function was sufficient to fit the
caffeine-induced Ca
release (Fig. 2A), implying that there is only one functional
store from which calcium is released. Therefore, these data suggested
that the mechanism of quantal release may have some other basis. As
shown in Fig. 2B, in the presence of thapsigargin but
in the absence of exogenous ATP caffeine not only released more
Ca
from the SR but also at a faster rate compared
with the caffeine response in the absence of thapsigargin. This
suggested that perhaps the SR Ca
-ATPase was active
during the first caffeine response, even though caffeine was applied in
the absence of ATP and the cells were washed with buffer A (60 s, no
ATP/GTP). Thus the graded release of Ca
at submaximal
caffeine concentrations might result from opposing effects of the SR
pumps and caffeine-activated Ca
release channels,
which may be operating to some extent even in an ATP-free medium,
perhaps because the SR Ca
-ATPase contains a tightly
bound ``ATP pool.''
To investigate this hypothesis 5 and
25 mM caffeine was applied incrementally to the same cell
before and after blocking the SR Ca-ATPase with
thapsigargin (Fig. 4). As shown in Fig. 4A,
caffeine at the lower concentration released more calcium from the SR
and at a faster rate after thapsigargin. In six cells treated in this
manner, the mean fluorescence ratio at rest was 2.07 ± 0.16 and
decreased to 1.50 ± 0.18 and 0.65 ± 0.04 in the presence
of 5 and 25 mM caffeine, respectively. The fluorescence ratio
increased to 1.79 ± 0.16 in the presence of ATP (buffer B) for 5
min. After thapsigargin (2 µM) incubation for 5 min the
resting ratio was 1.76 ± 0.17 and decreased to 0.92 ±
0.06 and 0.71 ± 0.04 in the presence of 5 and 25 mM caffeine, respectively (Fig. 4B). While the
fluorescence ratio recovered to a variable degree after initial
exposure to caffeine (range, 65-100%), the conversion of the
response to caffeine following thapsigargin from being decidedly graded
to largely all-or-none was not correlated with the extent of this
recovery (r
= 0.21). These data supported
our hypothesis that the SR Ca
-ATPase was active
during the first caffeine applications, even though ATP was absent from
the medium. However, after blocking the SR calcium pumps the stores
were still capable of releasing more calcium in the presence of a high
caffeine concentration. As noted in Fig. 2, thapsigargin was not
100% effective in blocking the SR calcium pumps at this concentration.
Therefore, a small fraction of the pumps might have remained active and
been able to establish an equilibrium between calcium influx and efflux
from internal stores, albeit at a lower store
[Ca
]. However, higher thapsigargin
concentrations were not used to avoid nonspecific membrane leakiness
seen at high concentrations(21) . The fluorescence ratio
throughout the 5-min incubation with thapsigargin remained constant,
indicating that the stores have a very low endogenous Ca
leak rate.
Figure 4: Effect of thapsigargin on the change in mag-fura 2 fluorescence ratio in response to caffeine at 5 and 25 mM in saponin-permeabilized smooth muscle cells. A, typical fluorescence ratio recorded from a cell stimulated with incremental caffeine concentrations (5 and 25 mM) before and after thapsigargin (2 µM, 5 min). B, mean and standard errors for the steady states in fluorescence ratio in 6 cells subject to the protocol in A.
An additional experiment was performed to test the
contribution of the SR Ca pumps to the quantal
release phenomenon in response to caffeine without resorting to the use
of pump inhibitors. In this series of experiments the putative
``ATP pool'' for SR Ca
pumps was depleted
by activation of these pumps at low caffeine concentration, and then
the cells were tested for graded Ca
release. For
this, cells were stimulated with 5 mM caffeine, put in buffer
A (no ATP) for 5 min, and then stimulated with 5 and 25 mM caffeine, during which time fluorescence ratio changes were
monitored (Fig. 5). The resting ratio was 2.15 ± 0.17 and
decreased to 1.41 ± 0.09 in the presence of 5 mM caffeine but in the absence of ATP. After buffer A (no ATP)
incubation for 5 min the ratio had increased to 1.70 ± 0.11 but
then decreased to 0.86 ± 0.03 and 0.79 ± 0.03 (n = 7) in the presence of 5 and 25 mM caffeine,
respectively (Fig. 5B). After the first caffeine
stimulus the cells were capable of calcium uptake as indicated by an
increased ratio, supporting the notion that a small amount of ATP
remained in the cell and was capable of fueling the SR calcium pump.
The second caffeine response released virtually all free Ca
from the SR, but higher caffeine concentration induced still a
small but significant release of Ca
. It is not clear
why this additional Ca
release occurred. It is
possible that mitochondria are capable of producing a small amount of
ATP and thus fuel the SR Ca
pump, or the mitochondria
may directly take up some Ca
and pass it on to the
SR. These data clearly indicate that the SR Ca
-ATPase
is active during the first caffeine application, partially returning
some of the released calcium back to the SR, ultimately resulting in a
steady state where not all Ca
has been released. In
addition, the second 5 mM caffeine application was capable of
lowering the final calcium concentration in the SR more than the first
stimulus, regardless of the initial luminal calcium concentration.
Figure 5: Effect of separated submaximal caffeine stimulation in the continued absence of ATP on mag-fura 2 fluorescence ratio in single saponin-permeabilized smooth muscle cells. A, typical fluorescence ratio recorded from a cell stimulated twice with 5 mM caffeine 5 min apart in the continued absence of ATP. B, mean and S.E. for the steady states in fluorescence ratio in 6 cells subject to the protocol in A. *, p < 0.05 compared with first 5 mM caffeine stimulus;**, p < 0.05 compared with second 5 mM caffeine stimulus.
In conclusion, graded increases in caffeine concentrations released
calcium from the SR in a quantal manner in isolated smooth muscle cells
permeabilized with saponin. These data cannot be reconciled with the
current three hypotheses to explain the underlying mechanism of quantal
release. Calcium release could be fit by a single exponential function,
which is inconsistent with the notion that 1) discrete stores have
different sensitivity to agonists and empty in an all or none fashion
or that 2) ryanodine receptors are heterogeneously distributed among
the stores. In addition, a second caffeine application of equal
magnitude was capable of releasing more calcium from the SR regardless
of the luminal calcium concentration, contrary to the notion that
partial emptying of stores results from an effect of luminal
[Ca] on the sensitivity of ryanodine
receptors. We have shown that the phenomenon of quantal release at
least in these cells in response to caffeine is largely due to the
activity of the SR Ca
-ATPase, which appears to return
a portion of the released Ca
back to the SR, even
though these applications were in the absence of ATP and the cells were
washed with an ATP-free buffer prior to these stimuli. Apparently, the
SR Ca
-ATPase is fueled by ATP, which is either
compartmentalized or bound to the SR membrane.
While the current
data may not be directly relevant to the mechanism underlying quantal
release of Ca in response to IP
or even
caffeine in other cells, they highlight that Ca
pumps
on internal stores are quite effective, at least acutely, in
resequestering Ca
as it leaves those stores even
after exogenous ATP has been removed and with Ca
buffers in the cytosol. Certainly this fact should be kept in
mind in interpreting quantal release phenomena. Recent work on other
single cells has shown that the ability to demonstrate quantal release
phenomena is a function of other experimental conditions as well
(Ca
buffering, Ca
indicator
affinity, and Ca
indicator location(
);
extent of fragmentation of the internal store
system(23, 24) ), suggesting that ``quantal
release'' may reflect the interplay of the Ca
release mechanism with other aspects of Ca
store function in the cell. While it may still be that quantal
release of Ca
at the cellular level is an inherent
property of the release mechanism per se, as suggested by the
work of Ferris et al.(22) , further carefully
controlled work on this intriguing phenomenon needs to be carried out.