©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Region-specific Activity of the Plasma Membrane CaPump and Delayed Activation of CaEntry Characterize the Polarized, Agonist-evoked CaSignals in Exocrine Cells (*)

Emil C. Toescu (§) , Ole H. Petersen

From the (1) Physiological Laboratory, Liverpool University, L69 3BX, Liverpool, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The initial release of Cafrom the intracellular Castores is followed by a second phase during which the agonist-dependent Caresponse becomes sensitive to the extracellular Ca, indicating the involvement of the plasma membrane (PM) Catransport systems. The time course of activation of these transport systems, which consist of both Caextrusion and Caentry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Caresponse, 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 Caextrusion system, and maximal activity was reached within less than 2 s. The rate of Caextrusion 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 Caentry 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 Castores and the activation of this entry pathway. By use of digital imaging data, the PM Catransport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Caentry pathways, no significant difference existed between these two regions. In contrast, the PM Capump 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 CaATPase might play an important role in the polarization of the Caresponse.


INTRODUCTION

In many cell types stimulation by agonists which enhance the production of InsP() initiate a complex succession of events which culminate with the release of Cafrom intracellular stores (1) . In exocrine cells, digital Caimaging 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 Casignal, which involves an initial increase in the lumenal pole followed by a spreading of a Catide 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 Carelease 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 Capools to the actions of the Careleasing agents (6, 7, 8, 9, 10) . However, we reported previously that the intracellular Cabuffers have also an important role in the regulation of the agonist-evoked Casignals (11, 12) . In a more general definition, these Cabuffers 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 Cafrom the cytosol. Among the latter, the PM CaATPase is an established contender, shown to extrude a substantial amount of Cafollowing the agonist-evoked Casignals (14) .

The initial release of Cais followed by a second phase of the Caresponse, sensitive to the manipulation of extracellular Ca. In the absence of extracellular Camaximal agonist stimulation still evokes an increase of [Ca], but only transient, due to the activation of the Caextrusion mechanisms, represented in pancreatic acinar cells mainly by the PM CaATPase (14, 15) . In the presence of extracellular Ca, the extrusion of Cais balanced by an entry of Cafrom 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 Caentry (16, 17) , point to the fact that the activation of the Caentry pathway is a consequence of the depletion of the intracellular Capools. Regarding the nature of this Capathway, several patch clamp studies (18, 19) identified a Ca-release-activated Cacurrent ( 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 Caentry 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 Carelease channel/receptor and the Caentry pathway mediated through an InsPreceptor (20) or through the cytoskeleton (17) . Other ``metabolic coupling'' models propose that the activation of the Caentry 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 ``Cainflux 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 Caentry 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 Capools and the activation of the Caentry pathway.

In the present study digital Caimaging technologies were used to investigate the spatial and temporal aspects of the activation of the plasma membrane Catransport 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 Caextrusion system is very different from that of the Caentry pathway, ( b) the activation of the Caentry pathway is significantly delayed in respect to the mobilization of Cafrom the intracellular pools, and ( c) the PM Capump display different patterns of activity in different regions of the cell, which could be an important mechanism in the polarization of the Casignal.


MATERIALS AND METHODS

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, MgCl1.1, 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 Catransport systems (28) , 1 m M Lawas added to the control solution (``lanthanum'' solution).

CaImaging and Data Analysis

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.

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 Caimaging 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 Caresponse varied from one experiment to another. To compare agonist-evoked Casignals in different experiments we have chosen to normalize the Casignal 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]).

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, in the presence of 2 m M ionomycin. The dissociation constant for fura 2 ( K) used for the calculation of the Cavalues 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).


RESULTS

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 Casignaling 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 Catransport systems can be ascertained. In the presence of maximal agonist stimulation and for the short duration (10 s) of these experiments, the InsP-activated Carelease 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 Caentry pathway (Fig. 1 A, process 2) is eliminated. We have shown recently (28) that 1 m M Laeffectively 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 Casignal (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 CaATPase. Similarly, subtraction of traces obtained in the absence of extracellular Cafrom traces obtained in control conditions ( trace B) will provide information about the behavior of the Caentry 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 Casignaling. Process 1 represents the release of Cafrom the intracellular pools; process 2, the Caentry pathway; process 3, the plasma membrane Caextrusion system; and process 4, the reuptake of Cainto the internal stores. B, schematic representation of the experimental traces. Information about the Caentry and/or Caextrusion 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 Caresponse.



Analysis of Plasma Membrane CaTransport Systems

The participation of the plasma membrane Catransport 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 Catransport system is activated very early during maximal agonist stimulation. Within 0.5 s the difference in [Ca]due to the PM CaATPase 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 Capump 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 Caextrusion 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 Lafrom those obtained in the absence of extracellular Ca(see Fig. 1). B, relationship between the apparent rate of Caextrusion 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 CaATPase 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 Caentry is significantly delayed, requiring more than 6 s for its activation. This time scale is to be contrasted with the time course of Carelease from the intracellular Capools following agonist stimulation (Fig. 3 B). Since Lainhibits all Catransport across the plasma membrane (28, 36) , the measured values of [Ca]in its presence reflect only the release from the intracellular Capools. 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 Cafrom the intracellular stores through the Carelease channels balanced by the reuptake into the stores through the endoplasmic reticulum CaATPase. 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 Carelease and precedes by 3-4 s the activation of the Caentry pathway.


Figure 3: Delayed activation of the PM Ca entry pathway. A, plot of the time course of activation of the PM Caentry pathway. Data are presented as the difference between the values recorded in control conditions and in the absence of extracellular Ca, when the Caentry pathway is inactive. B, the time course of the activation of the PM Caentry 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 CaTransport Activity during Polarized CaSignals

The use of Caimaging allows the analysis of changes in [Ca]in different regions of the cell. In many exocrine cells, the Casignal 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 Catransport in different regions of the pancreatic acinar cells.

Fig. 4A shows the mean increases in [Ca]recorded during the agonist-evoked Casignal in the lumenal pole of pancreatic acinar cells in the presence of Laor 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 Caextrusion 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 CaATPase 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 CaATPase maintains the same level of activity despite further increases of [Ca]. Using the same procedure, the activity of the PM CaATPase 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 Caextrusion 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 Caextrusion 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 Caextrusion 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 Caextrusion 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 Caextrusion activity is associated with further increases in [Ca]. The relationship between Caextrusion 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 Caabove the resting [Ca]value, the extrusion in the lumenal pole will account for a Cadifference 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 Caentry 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 Caextrusion system, there are no significant differences in the activation of the PM Caentry 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 Caentry (presented as the difference in [Ca]) at both poles of the cell: lumenal ( filled squares) and basal ( open squares).




DISCUSSION

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 Catransport systems, i.e. the extrusion and the entry pathway, is significantly different, with the Caentry pathway becoming active more than 5 s after the initiation of the Carelease from the intracellular Capools. Second, it is shown that the polarization of the Casignal, reported previously for several cell types (2, 3, 4, 5, 29) , is associated with significant differences in the activity of the PM Caextrusion system in two functionally distinct regions of the pancreatic acinar cells, the lumenal and the basal pole.

The question of the participation of the plasma membrane Catransport systems during the agonist-evoked Casignal is still debated. The entry of Cainto 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 Capools activates a Caentry pathway (16) . The molecular nature of this Caentry pathway and, more importantly, the mechanisms which activate it are the subject of intensive research.

In some cell types, the burst of [Ca]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 Cais an essential primer for the ensuing [Ca]increase. This primer Cainitially loads the intracellular pools and subsequently sensitize the Carelease process eventually determining the sudden release of Cafrom 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 Caentry appears a few seconds after the maximal release of Cafrom the stores. This delayed activation has also been suggested previously, in experiments using short 0Capulses 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 Cachannel (37) or the participation of a plasma membrane, InsP-activated Cachannel (38, 39, 40) , and suggests the existence of a metabolic step interposed between store depletion and Caentry 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) .

In pancreatic acinar cells, the main Caextrusion system is represented by the PM CaATPase since the participation of the Na/Caexchanger 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.

Analysis of the PM Caextrusion 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 Casignal (2, 4) . This polarization could be explained on the basis of differential sensitivity of intracellular Castores to the action of the Careleasing agents (6, 8, 10) . This hypothesis is supported by recent immunolocalization data showing a preferential localization of the InsPreceptor 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 Cabuffers induce significant changes in the pattern of Caoscillations (11) . In situ cytosolic Cabuffers have been invoked in a model explaining the characteristics of the interspike period (43) . We have also proposed that the status of intracellular Cabuffers play an important role in determining the specific pattern of Caoscillations independent of the nature of the agonist (12) . In this context, the PM Caextrusion system can be seen as an effective cytosolic Cabuffer. The different activities in the two poles of the pancreatic acinar cells might play an important role in the generation of the polarity of Casignal. Thus, even with a homogenous release of Caacross 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 CaATPase 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) .

In summary, the present study shows that the initiation of the agonist-evoked Casignal is associated with complex changes in the plasma membrane Catransport systems. The increase of [Ca]following the release of Cafrom the intracellular Castores rapidly (within 1 s) activates the PM Caextrusion 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 CaATPase might play an important role in the polarization of the Caresponse. The maximal ``effective'' depletion of the intracellular Capools does not trigger immediately the activation of the Caentry pathway, suggesting the existence of a metabolic step interposed between these two processes.


FOOTNOTES

*
The work was funded by grants from the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology, The School of Medicine, Edgbaston, Birmingham B15 2TT, UK.

The abbreviations used are: InsP, inositol 1,4,5-trisphosphate; InsP, inositol 1,3,4,5-tetrakiphosphate; PM, plasma membrane.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  2. Kasai, H., and Augustine, G. J. (1990) Nature 348, 735-738 [CrossRef][Medline] [Order article via Infotrieve]
  3. Rooney, T. A., Sass, E., and Thomas, A. P. (1990) J. Biol. Chem. 265, 10792-10796 [Abstract/Free Full Text]
  4. Toescu, E. C., Lawrie, A. M., Petersen, O. H., and Gallacher, D. V. (1992) EMBO J. 11, 1623-1629 [Abstract]
  5. Theler, J. M., Mollard, P., Guerineau, N., Vacher, P., Pralong, W. F., Schlegel, W., and Wolheim, C. B. (1992) J. Biol. Chem. 267, 18110-18117 [Abstract/Free Full Text]
  6. Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668 [Medline] [Order article via Infotrieve]
  7. Toescu, E. C., Gallacher, D. V., and Petersen, O. H. (1994) Biochem. J. 304, 313-316 [Medline] [Order article via Infotrieve]
  8. Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677 [Medline] [Order article via Infotrieve]
  9. Lawrie, A. M., Toescu, E. C., and Gallacher, D. V. (1993) Cell Calcium 14, 698-710 [Medline] [Order article via Infotrieve]
  10. van de Putt, F. H. M. M., De Pont, J. J. H. H. M., and Willems, P. H. G. M. (1994) J. Biol. Chem. 269, 12438-12443 [Abstract/Free Full Text]
  11. Petersen, C. C. H., Toescu, E. C., and Petersen, O. H. (1991) EMBO J. 10, 527-533 [Abstract]
  12. Toescu, E. C., Lawrie, A. M., Gallacher, D. V., and Petersen, O. H. (1993) J. Biol. Chem. 268, 18654-18658 [Abstract/Free Full Text]
  13. Kasai, H. (1993) Neurosci. Res. 16, 1-7 [Medline] [Order article via Infotrieve]
  14. Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572 [Abstract/Free Full Text]
  15. Muallem, S. (1989) Annu. Rev. Physiol. 51, 83-105 [CrossRef][Medline] [Order article via Infotrieve]
  16. Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624 [Medline] [Order article via Infotrieve]
  17. Putney, J. W., Jr., and Bird, G. S. J. (1993) Endocr. Rev. 14, 610-631 [Medline] [Order article via Infotrieve]
  18. Hoth, M., and Penner, R. (1992) Nature 355, 353-356 [CrossRef][Medline] [Order article via Infotrieve]
  19. Fasolato, C., Hoth, M., and Penner, R. (1993) J. Biol. Chem. 268, 20737-20740 [Abstract/Free Full Text]
  20. Irvine, R. F. (1992) FASEB J. 6, 3085-3091 [Abstract/Free Full Text]
  21. Alvarez, J., Montero, M., and Garcia-Sancho, J. (1992) FASEB J. 6, 786-792 [Abstract/Free Full Text]
  22. Xu, X., Star, R. A., Tortorici, G., and Muallem, S. (1994) J. Biol. Chem. 269, 12645-12653 [Abstract/Free Full Text]
  23. Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814 [CrossRef][Medline] [Order article via Infotrieve]
  24. Bird, G. S., and Putney, J. W. J. (1992) J. Biol. Chem. 268, 21486-21488 [Abstract/Free Full Text]
  25. Lee, K.-M., Toscas, K., and Villereal, M. L. (1993) J. Biol. Chem. 268, 9945-9948 [Abstract/Free Full Text]
  26. Llopis, J., Kass, G. E. N., Gahm, A., and Orrenius, S. (1992) Biochem. J. 284, 243-247 [Medline] [Order article via Infotrieve]
  27. Kass, G. E. N., Webb, D.-L., Chow, S. C., Llopis, J., and Berggren, P.-O. (1994) Biochem. J. 302, 5-9 [Medline] [Order article via Infotrieve]
  28. Toescu, E. C., and Petersen, O. H. (1994) Pfluegers Arch. 444, 325-331
  29. Elliot, A. C., Cairns, S. P., and Allen, D. G. (1992) Pfluegers Arch. 422, 245-252 [Medline] [Order article via Infotrieve]
  30. Tsien, R. W., and Tsien, R. Y. (1990) Annu. Rev. Cell Biol. 6, 715-716 [CrossRef]
  31. Iino, M., Yamazawa, T., Miyashita, Y., Endo, M., and Kasai, H. (1993) EMBO J. 12, 5287-5291 [Abstract]
  32. Friel, D. D., and Tsien, R. W. (1992) Neuron 8, 1109-1125 [Medline] [Order article via Infotrieve]
  33. Miyazaki, S., Yuzaki, M., Nakada, K., Shirakawa, H., Nakanishi, S., Nakade, S., and Mikoshiba, K. (1992) Science 257, 251-255 [Medline] [Order article via Infotrieve]
  34. Berridge, M. J. (1994) Biochem. J. 302, 545-550 [Medline] [Order article via Infotrieve]
  35. Goldbetter, A., Dupont, G., and Berridge, M. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1461-1465 [Abstract]
  36. Kwan, C.-Y., Takemura, H., Obie, J. F., Thastrup, O., and Putney, J. W. J. (1990) Am. J. Physiol. 258, C1006-C1015
  37. Felder, C., Poulter, M. O., and Wess, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 509-513 [Abstract]
  38. Kuno, M., and Gardner, P. (1987) Nature 326, 301-304 [CrossRef][Medline] [Order article via Infotrieve]
  39. Khan, A. A., Steiner, J. P., Kelin, M. G., Schneider, M. F., and Snyder, S. H. (1992) Science 249, 1166-1168
  40. Fadool, D. A., and Ache, B. W. (1992) Neuron 9, 907-918 [Medline] [Order article via Infotrieve]
  41. Koch, B. D., Faurot, G. F., Kopanitsa, M. V., and Swinney, D. C. (1994) Biochem. J. 302, 187-190 [Medline] [Order article via Infotrieve]
  42. Nathanson, M. H., Fallon, M. B., Padfield, P. J., and Maranto, A. R. (1994) J. Biol. Chem. 269, 4693-4696 [Abstract/Free Full Text]
  43. Petersen, C. C. H., Petersen, O. H., and Berridge, M. J. (1993) J. Biol. Chem. 268, 22262-22264 [Abstract/Free Full Text]
  44. Carafoli, E., and Stauffer, T. (1994) J. Neurobiol. 25, 312-324 [Medline] [Order article via Infotrieve]
  45. Enyedi, A., Filoteo, A. G., Gardos, G., and Penniston, J. T. (1991) J. Biol. Chem. 266, 8952-8956 [Abstract/Free Full Text]
  46. Strehler, E. E., Strehler-Page, M.-A., Vogel, G., and Carafoli, E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6908-6912 [Abstract]
  47. Kessler, F., Bennardini, F., Bachs, O., Serratosa, J., James, P., Caride, A. J., Gazzotti, P., Penniston, J. T., and Carafoli, E. (1990) J. Biol. Chem. 265, 16012-16019 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.