From the
The initial release of Ca
In many cell types stimulation by agonists which enhance the
production of InsP
The initial release of Ca
In the present study digital
Ca
The images were captured using a
Nikon Diaphot inverted microscope, an intensified charge coupled device
camera (Photonic Science Inc.) and recorded on a MagiCal station
(Applied Imaging, UK). Other details of the Ca
Fura-2
fluorescence was calibrated using the cells loaded in the normal way
and perfused subsequently either with 10 m
M EGTA or with 10
m
M Ca
Fig. 4A shows the mean increases in
[Ca
Two main conclusions emerge from the present results. The
first one is that, following maximal agonist stimulation, the time
course of activation of the two major PM Ca
The
question of the participation of the plasma membrane Ca
In some cell types, the burst of
[Ca
In pancreatic acinar
cells, the main Ca
Analysis
of the PM Ca
In summary, the present study shows that
the initiation of the agonist-evoked Ca
E. C. T. thanks Prof. O. H. Petersen for his
continuous support and interest during the stay in Liverpool and V. J.
T. for showing and discussing new perspectives.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
from the
intracellular Ca
stores is followed by a second phase
during which the agonist-dependent Ca
response
becomes sensitive to the extracellular Ca
, indicating
the involvement of the plasma membrane (PM) Ca
transport systems. The time course of activation of these
transport systems, which consist of both Ca
extrusion
and Ca
entry pathways, is not well established. To
investigate the participation of these processes during the
agonist-evoked Ca
response, isolated pancreatic
acinar cells were exposed to maximal concentrations of an inositol
1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10
µ
M) in different experimental conditions. Following the
increase of [Ca
]
,
there was an almost immediate activation of the PM Ca
extrusion system, and maximal activity was reached within less
than 2 s. The rate of Ca
extrusion was dependent on
the level of [Ca
]
,
with a steep activation at values just above the resting
[Ca
]
and reached a
plateau value at 700 n
M Ca
. In contrast, the
PM Ca
entry pathway was activated with a much slower
time course. There was also a delay of 3-4 s between the maximal
effective depletion of the intracellular Ca
stores
and the activation of this entry pathway. By use of digital imaging
data, the PM Ca
transport systems were also analyzed
independently in two regions of the cells, the lumenal and the basal
poles. With respect to the activation of the Ca
entry
pathways, no significant difference existed between these two regions.
In contrast, the PM Ca
pump displayed a different
pattern of activity in these regions. In the basal pole, the pump
activity was more sensitive to changes of
[Ca
]
and had a higher
maximal activity. Also, in the lumenal pole, the pump became saturated
at values of [Ca
]
around 700 n
M, whereas at the basal pole
[Ca
]
had a biphasic
effect on the pump activity, and higher
[Ca
]
inhibited the
pump. It is argued that these differences in sensitivity to the levels
of [Ca
]
and the
different relationship between
[Ca
]
and the rate of
extrusion at the two functional poles of the pancreatic acinar cells
indicate that the plasma membrane Ca
ATPase might play
an important role in the polarization of the Ca
response.
(
)
initiate a
complex succession of events which culminate with the release of
Ca
from intracellular stores
(1) . In exocrine
cells, digital Ca
imaging techniques revealed that
this release shows a spatial polarization which mirrors the functional
polarization characteristic for these cells
(2, 3, 4, 5, 6) . This spatial
organization of the Ca
signal, which involves an
initial increase in the lumenal pole followed by a spreading of a
Ca
tide toward the basolateral regions, can be evoked
either by agonist stimulation
(2, 4, 7) or by
direct intracellular perfusion of InsP
(6, 8) . We have shown recently that this
polarization is not due to a difference in the kinetic properties of
the Ca
release process in the two regions of the cell
(7) . An important functional mechanism underlying this
polarization is the heterogeneity in the sensitivity of the
intracellular Ca
pools to the actions of the
Ca
releasing agents
(6, 7, 8, 9, 10) . However, we
reported previously that the intracellular Ca
buffers
have also an important role in the regulation of the agonist-evoked
Ca
signals
(11, 12) . In a more
general definition, these Ca
buffers include not only
the cytosolic Ca
-binding proteins, already implicated
in a model of ``dynamic decoding''
(13) , but also the
systems involved in the removal of Ca
from the
cytosol. Among the latter, the PM Ca
ATPase is an
established contender, shown to extrude a substantial amount of
Ca
following the agonist-evoked Ca
signals
(14) .
is followed by a second phase of the Ca
response, sensitive to the manipulation of extracellular
Ca
. In the absence of extracellular Ca
maximal agonist stimulation still evokes an increase of
[Ca
]
, but only
transient, due to the activation of the Ca
extrusion
mechanisms, represented in pancreatic acinar cells mainly by the PM
Ca
ATPase
(14, 15) . In the presence of
extracellular Ca
, the extrusion of Ca
is balanced by an entry of Ca
from the external
medium, the two processes resulting in a maintained, steady-state
plateau of increased
[Ca
]
. Several lines of
evidence, brought together into the model of capacitative
Ca
entry
(16, 17) , point to the fact
that the activation of the Ca
entry pathway is a
consequence of the depletion of the intracellular Ca
pools. Regarding the nature of this Ca
pathway,
several patch clamp studies
(18, 19) identified a
Ca
-release-activated Ca
current
( I
). What is still very much a matter of debate
is the question of how the depletion of the stores is transduced into
the activation of the Ca
entry channel and numerous
hypotheses have been proposed to account for this process. These
include models of ``mechanical coupling'' involving direct
interactions between the InsP
-sensitive Ca
release channel/receptor and the Ca
entry
pathway mediated through an InsP
receptor
(20) or
through the cytoskeleton
(17) . Other ``metabolic
coupling'' models propose that the activation of the
Ca
entry pathway is the result of either ( a)
the stimulation of certain metabolic pathways, such as the cytochrome
P450
(21) or the NO/cGMP
(22) , ( b) the release
of a diffusible messenger (the ``Ca
influx
factor''
(23) ), or ( c) the activation of small
GTP-binding proteins
(19, 24) or tyrosine kinase(s)
(25) . The analysis is further complicated by the fact that in
some cell types, such as the hepatocytes, agonist stimulation activates
several Ca
entry pathways
(26, 27) .
An important parameter which could help in differentiating between
these various proposals is that of the temporal relationship between
the depletion of the Ca
pools and the activation of
the Ca
entry pathway.
imaging technologies were used to investigate the
spatial and temporal aspects of the activation of the plasma membrane
Ca
transport systems following the maximal
stimulation of the pancreatic acinar cells by an
InsP
-mobilizing agonist. The main conclusions of this study
are that ( a) the temporal activation of the Ca
extrusion system is very different from that of the
Ca
entry pathway, ( b) the activation of the
Ca
entry pathway is significantly delayed in respect
to the mobilization of Ca
from the intracellular
pools, and ( c) the PM Ca
pump display
different patterns of activity in different regions of the cell, which
could be an important mechanism in the polarization of the
Ca
signal.
Preparation of Cells
Individual cells and small
clusters of acinar cells were obtained from isolated mouse pancreata by
enzymatic dispersion in a water bath at 37 °C as described
previously
(4) . Briefly, the intact glands were injected with a
collagenase solution (200 units/ml, Worthington), incubated for
7-15 min, and finally manually agitated to yield the isolated
cell preparation. Throughout the preparation procedure and subsequently
in some experiments, a buffer solution (``control'' solution)
containing (m
M): NaCl 140, KCl 4.7, CaCl1.1,
MgCl
1.1, glucose 10, and HEPES 10 (pH 7.2 adjusted with
NaOH) with an osmolality of 295 mOsm/liter was used. For some
experiments a ``0 Ca'' solution was used which consisted of a
nominally Ca
-free control solution to which 0.5
m
M EGTA was added. To inhibit all the plasma membrane
Ca
transport systems
(28) , 1 m
M La
was added to the control solution
(``lanthanum'' solution).
Ca
Cells were loaded with 1 µ
M fura-2-AM for
30 min at room temperature, washed twice, and used within 3-4 h.
During experiments, the cells, placed on a glass coverslip attached to
an open perfusion chamber, were continuously perifused from a
gravity-fed perifusion system.
Imaging and Data
Analysis
imaging were described previously
(9) . The fluorescence
signals were captured, at video-frame rate (0.16-s intervals between
two true ratio images) with a 20
magnification objective. In
each field, comprising several cells, those cells responding to agonist
were analyzed by calculating the mean
[Ca
]
value within
areas defined with a light-sensitive pen on a bright field image
showing the morphology of cells. The areas used for these measurements
were of a similar size to those used by us previously
(4, 9) . Due to biological variability and to the
inherently variable geometry of our perifusion method, the lag time
between the start of agonist application and the initiation of
Ca
response varied from one experiment to another. To
compare agonist-evoked Ca
signals in different
experiments we have chosen to normalize the Ca
signal
to the first time point of the
[Ca
]
increase (defined
as the first point of increase of
[Ca
]
which is followed
by two consecutive higher values of
[Ca
]
).
, in the presence of 2 m
M ionomycin. The dissociation constant for fura 2
( K
) used for the calculation of the
Ca
values was 224 n
M.
Other Materials
Chemicals were purchased from
Merck-BDH (UK) and Sigma (UK) except fura-2 which was obtained from
Novo-Calbiochem (UK).
General Method of Analysis
Fig. 1A depicts schematically the major Catransport
processes which participate in establishing the value of
[Ca
]
during the
agonist-evoked, InsP
-mediated Ca
signaling in nonexcitable cells. Clearly, in such studies which
measure the resultant
[Ca
]
, the direct and
separate assessment of the participation of each process to the overall
[Ca
]
value is not
possible. Nevertheless, by using certain experimental conditions and by
performing simple algebraic additions as shown in
Fig. 1B, the relative participation of the plasma
membrane Ca
transport systems can be ascertained. In
the presence of maximal agonist stimulation and for the short duration
(10 s) of these experiments, the InsP
-activated
Ca
release channel (Fig. 1 A, process
1) is assumed to be continuously activated. In the absence of
extracellular Ca
(Fig. 1 B, trace
C), the participation of the capacitative Ca
entry pathway (Fig. 1 A, process 2) is
eliminated. We have shown recently
(28) that 1 m
M La
effectively seals the cell with respect to
extracellular Ca
, blocking both the entry and the
extrusion of Ca
(Fig. 1 A,
processes 2 and 3, respectively) and results in an
increased Ca
signal
(28) (Fig. 1 C). As shown in Fig. 1 B, the
difference between traces obtained in the presence of La
( trace A) and those obtained in the absence of
extracellular Ca
( trace C) are due only to
the activity of the plasma membrane Ca
ATPase.
Similarly, subtraction of traces obtained in the absence of
extracellular Ca
from traces obtained in control
conditions ( trace B) will provide information about the
behavior of the Ca
entry pathway. Fig. 1 C shows the actual experimental (mean) values, measured across the
whole cell and normalized for the first value of increased
[Ca
]
as detailed under
``Materials and Methods.''
Figure 1:
Principles of analysis. A,
schematic representation of the Catransport systems
involved during the agonist-evoked Ca
signaling.
Process 1 represents the release of Ca
from
the intracellular pools; process 2, the Ca
entry pathway; process 3, the plasma membrane
Ca
extrusion system; and process 4, the
reuptake of Ca
into the internal stores. B,
schematic representation of the experimental traces. Information about
the Ca
entry and/or Ca
extrusion
pathways can be derived by simple algebraic summation of individual
processes (see text for further details). C, actual
experimental traces of [Ca
] following
maximal stimulation with acetylcholine (10 µ
M). The values
plotted represent mean values for each time point derived from:
Lanthanum, four separate experiments (31 cells);
Control, six separate experiments (65 cells); 0 Ca,
five experiments (54 cells). Time t = 0 represents the
time of the first increase in [Ca
] for each
individual cell and defined under ``Materials and Methods.''
For clarity, the error bars were omitted (for each time point they
represent 10-20% of the mean value). The inset shows, on
an expanded time scale, the first 2 s of the Ca
response.
Analysis of Plasma Membrane Ca
The participation of the plasma membrane
CaTransport Systems
transport system was assessed for each time point,
as the difference between the
[Ca
]
measured in the
presence of extracellular La
(1 m
M) and in
the absence of extracellular Ca
(Fig. 1 B). In agreement with our previous
suggestion
(28) , it appears that this Ca
transport system is activated very early during maximal agonist
stimulation. Within 0.5 s the difference in
[Ca
]
due to the PM
Ca
ATPase is manifest, and it reaches a maximal level
in less than 2 s (Fig. 2 A), by which time it accounts for a
difference of 350 n
M Ca
. Throughout the rest
of the stimulation the Ca
pump remains active. An
important property of this transport system is illustrated in Fig.
2 B which shows the relationship between the apparent extrusion
rate, calculated from data shown in Fig. 2 A as the
difference ( d) in
[Ca
]
values per unit
of time
( d[Ca
]
/ dt),
and the values of
[Ca
]
. The noteworthy
feature of this relationship is its steepness: at values of
[Ca
]
over 200
n
M, even small changes in the value of
[Ca
]
evoke big
increases in the apparent rate of extrusion. At
[Ca
]
values around 600
n
M, the apparent rate is maximal (370 n
M Ca
/s). Further increases of
[Ca
]
do not determine
any significant additional activation.
Figure 2:
Participation of the Ca
extrusion system during the agonist-evoked Ca
signal.
A, plot of the time course of activation of the PM
Ca
extrusion system. Data are presented as the
difference in [Ca
] due to the activation of
this system and was derived by subtracting, for each individual time
point, the [Ca
] values recorded in the
presence of La
from those obtained in the absence of
extracellular Ca
(see Fig. 1). B,
relationship between the apparent rate of Ca
extrusion and the value of [Ca
] at
which that rate was recorded. The apparent extrusion rate (ordinate)
was calculated as the difference in mean
[Ca
] values between two consecutive time
points ( d[Ca
]) shown in Panel
A and divided by the respective time interval ( dt). These
values for the extrusion rate are plotted against the actual
[Ca
] levels (on the abscissa) at
which the rate was calculated. The values plotted on the abscissa are
the values of [Ca
] recorded in the absence
of extracellular Ca
, condition in which the PM
Ca
ATPase is active.
The activation of the
Caentry pathway can be derived from the comparison
between control experiments and those performed in the absence of
extracellular Ca
(see Fig. 1 B). It is
apparent from Fig. 1 C that the values of
[Ca
]
measured in the
first seconds after the beginning of
[Ca
]
increase are, in
pancreatic acinar cells, little affected by the absence of
extracellular Ca
, and the two curves are almost
superimposable. As shown in Fig. 3 A the participation of
the Ca
entry is significantly delayed, requiring more
than 6 s for its activation. This time scale is to be contrasted with
the time course of Ca
release from the intracellular
Ca
pools following agonist stimulation (Fig.
3 B). Since La
inhibits all Ca
transport across the plasma membrane
(28, 36) ,
the measured values of [Ca
]
in its presence reflect only the release from the intracellular
Ca
pools. The trace plotted in Fig. 3 B shows that, by 3 s, the
[Ca
]
reached a
steady-state level, maintained afterward. This steady-state level is
the result of the maximal efflux of Ca
from the
intracellular stores through the Ca
release channels
balanced by the reuptake into the stores through the endoplasmic
reticulum Ca
ATPase. As such, it represents a level of
maximal ``effective'' depletion of the pools, which is thus
completed by 3 s after the initiation of the Ca
release and precedes by 3-4 s the activation of the
Ca
entry pathway.
Figure 3:
Delayed activation of the PM
Ca entry pathway. A, plot of the time course
of activation of the PM Ca
entry pathway. Data are
presented as the difference between the values recorded in control
conditions and in the absence of extracellular Ca
,
when the Ca
entry pathway is inactive. B,
the time course of the activation of the PM Ca
entry
pathway ( filled squares, left ordinate) is plotted
together with the values of [Ca
] recorded
in the presence of La
(1 m
M) ( line
trace, right ordinate).
Regional Analysis of Ca
The use
of CaTransport
Activity during Polarized Ca
Signals
imaging allows the analysis of changes in
[Ca
]
in different
regions of the cell. In many exocrine cells, the Ca
signal is initially localized to the lumenal pole from where,
0.3-0.5 s later, it spreads toward the basolateral pole. Using a
similar methodology, we investigated the pattern of activation and the
apparent kinetic properties of the PM Ca
transport in
different regions of the pancreatic acinar cells.
]
recorded during
the agonist-evoked Ca
signal in the lumenal pole of
pancreatic acinar cells in the presence of La
or
absence of extracellular Ca
, respectively. As in
previous figures the data were normalized to the first increase in
[Ca
]
. From these
results, the time course of the activation of the PM Ca
extrusion system at the lumenal pole was calculated and shown in
Fig. 4B. In this figure the extrusion activity is
plotted together with the ``reference''
[Ca
]
trace ( i.e. that recorded in the absence of extracellular Ca
, when
the PM Ca
ATPase is active). The increase of
[Ca
]
rapidly activates
the extrusion system, and its activity is maximal in less than 2 s, by
which time it accounts for a difference of about 200 n
M Ca
. It can also be seen from Fig. 4 B that the lumenal PM Ca
ATPase maintains the same
level of activity despite further increases of
[Ca
]
. Using the same
procedure, the activity of the PM Ca
ATPase in the
basal pole of the cells was assessed. These values are plotted in Fig.
4 C together with the corresponding reference
[Ca
]
trace in that
region of the cell. To allow a direct comparison of the activation
curves between the two regions, the initial time point in this graph is
represented by the first increase of
[Ca
]
not in
this region, but in the lumenal pole. In the present set of data, as in
previously published reports
(2, 4, 6, 8) , the increase of
[Ca
]
in the lumenal
pole precedes that in the basal pole by 0.3-0.4 s. In striking
contrast to the activity of the Ca
extrusion system
in the lumenal pole (Fig. 4 B), in the basal pole of the
pancreatic acinar cells the activity of the pump, after being maximally
stimulated within 1.5 s, is inhibited despite further increases of
[Ca
]
.
Figure 4:
Regional analysis of the activity of the
PM Ca extrusion. A, plot of the
[Ca
] values recorded in the lumenal pole.
The two traces show mean [Ca
] values from
cells incubated in the presence of 1 m
M La
( closed squares) or absence of extracellular
Ca
( open squares). For this analysis some
experiments were paired (performed successively, in the same day, with
cells derived from the same animal). The data shown were derived from:
Lanthanum, 7 experiments (27 cells); 0 Ca, eight
experiments (33 cells). B, plot of the time course of
Ca
extrusion at the lumenal pole ( filled
squares, left ordinate), presented as the difference in
[Ca
] between the two traces shown in
Panel A and the time course of
[Ca
] response ( line trace,
right ordinate) recorded at the same pole in the absence of
extracellular Ca
. C, same data presentation
as in Panel B but with the corresponding values for the basal
pole of the cells.
A direct
comparison of the time course of the activation of the Caextrusion systems in the two regions of the cell is presented in
Fig. 5A which illustrates better the important
differences between these two activities. The apparent rate of
Ca
extrusion activation in the basal pole of the cell
is almost twice as great as that seen in the lumenal pole. This
increased rate of activity is associated also with a greater maximal
activity. Finally, whereas the apparent activity of the PM
Ca
extrusion system in the lumenal pole remains
largely constant, in the basal pole it decreases immediately following
its peak of activation. As seen in Fig. 4 B, this
decrease of Ca
extrusion activity is associated with
further increases in
[Ca
]
. The relationship
between Ca
extrusion and
[Ca
]
for the two
regions of the cell is presented in Fig. 5 B. Both the
lumenal and the basal extrusion systems are activated by an increase of
[Ca
]
, but the
activation in the basal pole is much more sensitive to changes in
[Ca
]
. Thus, for
increases of 100 n
M Ca
above the resting
[Ca
]
value, the
extrusion in the lumenal pole will account for a Ca
difference of only 40 n
M Ca
, whereas
in the basal pole the difference is 150 n
M Ca
. In both regions the extrusion systems reach
a maximum activity at [Ca
]
around 0.7 µ
M, but the maximum value at the basal
pole is higher than that in the lumenal pole. As discussed before, at
higher values of [Ca
]
the extrusion in the two regions behave differently in respect to
[Ca
]
: whereas in the
lumenal region the activity remains constant, in the basal pole further
increases of [Ca
]
are
associated with a decrease of extrusion, indicating an inhibitory
effect of [Ca
]
.
Figure 5:
Differences in the activity of the PM
CaATPase at the two poles of the pancreatic acinar cells. A,
direct comparison of the extrusion activity at the two poles: lumenal
( closed squares) and basal ( open squares).
B, plot of the PM CaATPase apparent extrusion rate (calculated
as described in legend to Fig. 2, Panel B) at the two poles of
the cell against the values of [Ca]. The
values plotted on the abscissa are the values of
[Ca
] recorded in the absence of
extracellular Ca
, a condition in which the PM
CaATPase is active.
Next, the activation of the Caentry pathway was
analyzed in a similar manner. Fig. 6 A shows the mean
[Ca
]
traces recorded
in the lumenal pole in control conditions and in the absence of
extracellular Ca
. From these traces the time course
of Ca
entry activation was calculated and is shown in
Fig. 6B ( filled squares). Fig. 6 B also shows that, in contrast to the activity of the PM
Ca
extrusion system, there are no significant
differences in the activation of the PM Ca
entry
pathway between the lumenal and basal poles of the pancreatic acinar
cells.
Figure 6:
Regional analysis of the activity of the
PM Ca entry pathway. A, plot of the
[Ca
] values recorded in the lumenal pole.
The two traces show mean [Ca
] values from
cell incubated in Control conditions ( closed squares) or
absence of intracellular Ca
( open squares).
For this analysis, some experiments were paired (in the sense described
in the legend to Fig. 4), and the data shown were derived from:
Control, seven experiments (52 cells); 0 Ca, six
experiments (29 cells). B, plot of Ca
entry
(presented as the difference in [Ca
]) at
both poles of the cell: lumenal ( filled squares) and basal
( open squares).
transport
systems, i.e. the extrusion and the entry pathway, is
significantly different, with the Ca
entry pathway
becoming active more than 5 s after the initiation of the
Ca
release from the intracellular Ca
pools. Second, it is shown that the polarization of the
Ca
signal, reported previously for several cell types
(2, 3, 4, 5, 29) , is associated
with significant differences in the activity of the PM Ca
extrusion system in two functionally distinct regions of the
pancreatic acinar cells, the lumenal and the basal pole.
transport systems during the agonist-evoked Ca
signal is still debated. The entry of Ca
into
cells along a strong electrochemical gradient is regulated by a variety
of mechanisms
(30) . In electrically nonexcitable cells a
salient feature of this process is that the depletion of the
intracellular Ca
pools activates a Ca
entry pathway
(16) . The molecular nature of this
Ca
entry pathway and, more importantly, the
mechanisms which activate it are the subject of intensive research.
]
increase during
threshold or maximal stimulation is preceded by a slow pacemaker
elevation of [Ca
]
(31, 32, 33) . Recently, it has been
proposed that an important component of this phase is the entry of
external Ca
(34) . This view extends a
previous model
(1, 35) in which it is proposed that an
influx of external Ca
is an essential primer for the
ensuing [Ca
]
increase.
This primer Ca
initially loads the intracellular
pools and subsequently sensitize the Ca
release
process eventually determining the sudden release of Ca
from the endoplasmic reticulum. Implicit in this model is the
fact that the pools are initially depleted of Ca
. The
situation in the pancreatic acinar cells appears somewhat different.
Stimulation of cells in the presence of lanthanum at a concentration
which effectively seals these cells from the extracellular
Ca
(28, 36) evokes an increase of
[Ca
]
which is more
rapid and of higher amplitude than in the absence of extracellular
Ca
(Fig. 1)
(28) indicating that the
InsP
-sensitive stores are fully
Ca
-loaded. In addition, treatment with thapsigargin
evokes, even in the absence of extracellular Ca
,
increases of [Ca
]
.
Finally, as shown in Fig. 3, the activation of Ca
entry appears a few seconds after the maximal release of
Ca
from the stores. This delayed activation has also
been suggested previously, in experiments using short 0Ca
pulses during the increase of
[Ca
]
following
thapsigargin exposure
(28) . The delay in Fig. 3indicates
another important feature: the entry is not activated directly as a
consequence of pool depletion. The length of this delay (3-5 s)
exclude, at least for this cell type, a number activation mechanisms,
including a direct receptor-regulated Ca
channel
(37) or the participation of a plasma membrane,
InsP
-activated Ca
channel
(38, 39, 40) , and suggests the existence of a
metabolic step interposed between store depletion and Ca
entry activation. The nature of this process is yet unclear. One
proposal involves the cytochrome P450
(21) , but more recent
evidence seem to argue strongly against it ( e.g. see Ref. 41).
Other possibilities include the (paracrine) mediation by a
Ca
-influx factor
(23) or the participation of
small G-proteins
(19, 24) .
extrusion system is represented by
the PM Ca
ATPase since the participation of the
Na
/Ca
exchanger is minimal
(14, 15) . The present data show that this system, which
is extremely powerful
(14) , is rapidly activated following
agonist stimulation, in a manner dependent on
[Ca
]
(Fig. 2 B). An important feature of this dependence
is its steepness: even small increases of
[Ca
]
above the resting
values induce significant increases in the pump activity.
extrusion in different regions of the
cell revealed that the apparent activity of the pump is different.
Pancreatic acinar cells, like many other cell types, display a
polarization of the Ca
signal
(2, 4) .
This polarization could be explained on the basis of differential
sensitivity of intracellular Ca
stores to the action
of the Ca
releasing agents
(6, 8, 10) . This hypothesis is supported by
recent immunolocalization data showing a preferential localization of
the InsP
receptor type 3 to the lumenal pole
(42) .
An alternative mechanism, not exclusive of the first, is the existence
of a differential buffering in different regions of the cell.
Intracellular addition of mobile, high capacity, low affinity
Ca
buffers induce significant changes in the pattern
of Ca
oscillations
(11) . In situ cytosolic Ca
buffers have been invoked in a
model explaining the characteristics of the interspike period
(43) . We have also proposed that the status of intracellular
Ca
buffers play an important role in determining the
specific pattern of Ca
oscillations independent of
the nature of the agonist
(12) . In this context, the PM
Ca
extrusion system can be seen as an effective
cytosolic Ca
buffer. The different activities in the
two poles of the pancreatic acinar cells might play an important role
in the generation of the polarity of Ca
signal. Thus,
even with a homogenous release of Ca
across the
cytosol, the more rapid activation of the pump and its higher level of
activity in the basal pole would explain the delay in the rise of
[Ca
]
at this pole.
Recent molecular biology studies showed that the generic PM
Ca
ATPase is in fact a large multigene family (PMCA
family)
(44) . To date, little is known about the differences in
functional properties between different isoforms, but the few data
available on this subject point to such a possibility. Different
isoforms can show significant differences in their affinity for
calmodulin
(45) or in the pattern of phosphorylation
(46) . It is possible that these isoforms might display a
differential distribution within the cell, as it has been shown in
hepatocytes
(47) .
signal is
associated with complex changes in the plasma membrane Ca
transport systems. The increase of
[Ca
]
following the
release of Ca
from the intracellular Ca
stores rapidly (within 1 s) activates the PM Ca
extrusion system. The reported differences in the sensitivity to
the levels of [Ca
]
and
the different relationships between
[Ca
]
and the rate of
extrusion at the two functional poles of the pancreatic acinar cells
(lumenal and basal) indicate that the plasma membrane
Ca
ATPase might play an important role in the
polarization of the Ca
response. The maximal
``effective'' depletion of the intracellular Ca
pools does not trigger immediately the activation of the
Ca
entry pathway, suggesting the existence of a
metabolic step interposed between these two processes.
, inositol 1,4,5-trisphosphate; InsP
,
inositol 1,3,4,5-tetrakiphosphate; PM, plasma membrane.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.