Spatial Aspects of Ca²⁺Signalling in Pancreatic Acinar Cells

Exocrine glands (such as pancreas or lacrimal) share a common morphology, with a primary fluid secretion from distally located acinar cells modified by proximally located duct cells. The Ca²⁺ signal observed in the acinar cells regulates the secretion of fluid and enzymes (Petersen, 1992). Fluid secretion is dependent on the opening of a Ca²⁺-dependent chloride conductance thought to be located in the luminal plasma membrane (Petersen and Gallacher, 1988). Enzyme secretion is via the Ca²⁺-dependent exocytosis of secretory zymogen granules on the luminal membrane, releasing the granule contents into the duct. The two main agonists in pancreatic acinar cells that promote an increase in [Ca²⁺]_i are acetylcholine (ACh) and cholecystokinin (CCK). ACh is released at nerve terminals and CCK acts as a circulating hormone (Petersen, 1992). ACh and CCK, at high concentrations, couple to a different set of G-proteins (Schnefel et al. 1988), stimulate phospholipase C (PLC) and both produce increases in InsP₃ (Matozaki et al. 1990; Rowley et al. 1990). Evidence obtained from microelectrode studies and from the injection of fluorescent markers, such as Lucifer Yellow (Petersen and Findlay, 1987), has shown that individual pancreatic acinar cells are grouped together and are joined by tight junctions into larger multicellular units. Within these units, some cells are thought not to possess secretagogue receptors but, nevertheless, Ca²⁺ increases are observed in these cells, possibly due to diffusion of Ca²⁺ or InsP₃ from neighbouring acinar cells (Dissing et al. 1993). These larger units of cells are likely to be of importance in the physiological control of secretion.

The [Ca²⁺]_i responses of acinar cells to agonists can be categorized into three distinct types. First, at supramaximal agonist concentrations a sustained elevation in [Ca²⁺]_i is observed. In the majority of reports, this [Ca²⁺]_i rise is initiated in the secretory pole of the cell and then spreads as a wave towards the basal pole (Kasai and Augustine, 1990). Second, at lower agonist concentrations, oscillations in [Ca²⁺]_i are recorded. ACh and CCK elicit patterns of oscillations that are different and characteristic for each agonist (Petersen et al. 1991a). Third, at lower and physiological agonist concentrations (Forster and Dockray, 1992), the combination of whole-cell patch-clamp and digital Ca²⁺ imaging has revealed local repetitive elevations of [Ca²⁺]_i that occur only in the secretory pole of the cell and do not spread to the basal pole (Thorn et al. 1993a).

Ca²⁺ oscillations have been proposed to provide a digitally encoded transduction of the stimulus strength (Berridge, 1993). It is still not known whether oscillations are a physiologically important mechanism in acinar cells. It can be said that concentrations of CCK sufficient to promote in vivo secretion always elicit oscillations in vitro. However, there are a number of possible problems that arise as a result of our normal experimental protocols. For example, intercellular communication is lost in the single-cell preparations normally used. This could be an important difference between experiments that show oscillations and the situation in vivo. Single-cell studies will continue to provide insights into Ca²⁺ signalling while questions of physiological relevance will have to be addressed by different experimental approaches.

A sustained secretory response in acinar cells is ultimately dependent on extracellular [Ca²⁺] (Douglas and Poisner, 1963). However, the initial response to high agonist concentrations is known to be the result of the rapid liberation of Ca²⁺ into the cytoplasm from Ca²⁺ stores within the cell (Nielsen and Petersen, 1972). Recordings of Ca²⁺-dependent currents have suggested that the [Ca²⁺]_i rise is not homogeneous (Osipchuck et al. 1990) but,with the advent of digital Ca²⁺ imaging, this has been most elegantly demonstrated by Kasai and Augustine (1990).

Kasai and Augustine (1990) determined the secretory pole of the pancreatic acinar cell as the locus for the initiation of the Ca²⁺ response to supramaximal stimulation, which then spread as a propagated wave across the cell. The secretory pole in acinar cells is easily identified as the region containing the dark (under phase contrast) zymogen granules. These findings have been reproduced by Toescu et al. (1992a) and Elliot et al. (1992) and extended to include a similar pattern for the Ca²⁺ signal evoked by agonists in acinar cells derived from the mouse lacrimal gland. In contrast, Dissing and collaborators have consistently found no evidence for a localized initiation of the Ca²⁺ signal (Dissing et al. 1990) and no satisfactory explanation has been found to reconcile the different findings. Kasai and Augustine (1990) proposed, from their data, a region of high InsP₃ sensitivity located in the secretory pole of the cell to explain the site of initiation of the Ca²⁺ signal. The spreading of the Ca²⁺ wave to the basal pole is thought to be by a propagated mechanism of Ca²⁺-induced Ca²⁺ release (CICR). Ca²⁺ waves in general have been proposed to be due to an autocatalytic process such as CICR (Lechleiter and Clapham, 1992). In this way, Ca²⁺ release from one site acts on a neighbouring Ca²⁺-sensitive site to promote further Ca²⁺ release and leads to the propagation of a spreading Ca²⁺ wave. In the pancreatic acinar cells, wave velocity has been studied using confocal microscopy (Nathanson et al. 1992) and was found to increase with increasing agonist concentrations, but was slowed by caffeine and by a high concentration of ryanodine. These data were interpreted in terms of the presence of ryanodine receptors in the basal pole of the cell that propagate the Ca²⁺ wave. However, it has recently been shown in our laboratory that caffeine can inhibit the production of InsP₃ (Toescu et al. 1992b) and it may, in fact, act directly to inhibit the InsP₃ receptor (Parker and Ivorra, 1991). Therefore, the effects observed by Nathanson et al. (1992) do not provide unequivocal evidence of wave spreading on a ryanodine receptor.

Ca²⁺ oscillations in electrically nonexcitable cells were first demonstrated in hepatocytes (Woods et al. 1986). In acinar cells, the bulk of our knowledge of Ca²⁺ oscillations has been provided through the whole-cell patch-clamp technique (Petersen, 1992). Early patch-clamp studies demonstrated Ca²⁺-dependent currents (Maruyama and Petersen, 1982a; Marty et al. 1984) in the plasma membrane of acinar cells. Monitoring of these Ca²⁺-dependent conductance pathways has been used to gave an indirect measure of the Ca²⁺ signal.

There are three types of Ca²⁺-dependent currents in the plasma membrane of most acinar cells, namely Cl⁻, K⁺ and nonselective cation channels (Petersen, 1992). Exceptionally, the rodent pancreas does not possess a Ca²⁺-dependent K⁺ channel, although there is recent evidence for a voltage-dependent K⁺ current (Thorn, 1993). Single-channel studies of the nonselective cation channel (equally permeable to Na⁺ and K⁺) demonstrated activation by cholecystokinin via an intracellular elevation of Ca²⁺ (Maruyama and Petersen, 1982a,b). The Cl⁻channel has been directly demonstrated in the lacrimal acinar cell (Marty et al. 1984) and there is considerable evidence from whole-cell recording studies of Cl⁻current activation during agonist stimulation (Wakui et al. 1989). In the pancreas, the Cl⁻current has been demonstrated to be more sensitive to [Ca²⁺]_i than the nonselective cation current (Randriamampita et al. 1988). This, and evidence that [ATP]_i blocked the nonselective cation channel (Suzuki and Petersen, 1988), led to the idea that it was not activated by physiological agonist concentrations. Recently, this conclusion has been shown to be incorrect. We now know that both the Cl⁻and the nonselective cation currents are activated by physiological concentrations of agonist (Thorn and Petersen, 1992, 1993a).

The validity of using Ca²⁺-dependent current activation as a measurement of changes in [Ca²⁺]_i has been demonstrated by dual recording of currents and [Ca²⁺]_i using whole-cell patch-clamp and photometric Ca²⁺ determination (Osipchuk et al. 1990). In these experiments, higher agonist concentrations elicited oscillations with simultaneous rises in [Ca²⁺]_i and increases in Ca²⁺-dependent conductance.

The agonist-evoked oscillatory responses have a characteristic temporal pattern dependent on the agonist used and the concentration of agonist (Petersen et al. 1991a; Yule et al. 1991). With just suprathreshold CCK concentrations, short-lasting spikes of Ca²⁺-dependent current (approximately 2s duration) are observed that are interspersed with broad transient current activation (approximately 30s duration) (Fig. 1A). At just suprathreshold concentrations of ACh, trains of short-lasting spikes of Ca²⁺-dependent current are observed, and at higher ACh concentrations the Ca²⁺ signal develops into sinusoidal oscillations (Fig. 1B). At higher CCK concentrations, the slow transients increase in frequency and the short spikes decrease in frequency (Petersen et al. 1991a). These oscillatory patterns probably reflect the activation of different sets of G-proteins associated with stimulation by ACh or CCK (Schnefel et al. 1988).

Experiments employing patch-clamp and simultaneous single-cell photon counting (Osipchuck et al. 1990) have been used to identify the response to agonists simultaneously recorded from the Ca²⁺-dependent currents and [Ca²⁺]_i. As mentioned previously, at higher agonist concentrations, [Ca²⁺]_i increases and Ca²⁺-dependent current activation occurred simultaneously. However, simultaneous activation of currents and rises in [Ca²⁺]_i did not always occur. The trains of short-lasting spikes of Ca²⁺-dependent current observed in response to low ACh concentrations did not induce any rise in the average cellular Ca²⁺ signal measured using photon counting. This was interpreted as an indication that the short-lasting current spikes were due either to a local rise in [Ca²⁺]_i, restricted to one region of the cell, or possibly to a rise in [Ca²⁺]_i in the subplasmalemmal region (Petersen et al. 1991a). Such discrete [Ca²⁺]_i changes would not be picked up in the average [Ca²⁺]_i signal. Clearly, to obtain data to test the hypothesis of a localized Ca²⁺ signal we require spatial information of the [Ca²⁺]_i response and so we have turned to combining patch-clamp with digital Ca²⁺ imaging techniques.

There are a number of important advantages in using whole-cell patch-clamp techniques for Ca²⁺ measurement when compared with the usual methods of dye-loading with acetoxymethyl esters. First, the patch pipette can be used to fill the cell with Fura-2 and thus to avoid the problems of compartmentalization associated with loading the cells with the acetoxymethyl esters (Connor, 1993). Second, it also enables us to control the intracellular buffering directly because we know the concentration of Fura-2 in the patch pipette. Ca²⁺ buffering has a significant influence on the temporal aspects of Ca²⁺ signalling (Neher and Augustine, 1992). In our experiments, it was important to keep the Fura-2 buffering as low as possible (200–400 μmol l⁻¹) consistent with obtaining good images at near video rates of capture.

We focused our interest on the localization of the [Ca²⁺]_i increase that gives rise to the short-lasting agonist-evoked Ca²⁺-dependent current spikes (Thorn et al. 1993a). Our experiments in the pancreas demonstrated repetitive [Ca²⁺]_i spikes restricted to the secretory pole region of the cell. In the case of low concentrations of CCK (5–30pmol l⁻¹), the restricted [Ca²⁺]_i spikes often preceded a [Ca²⁺]_i wave that spread across the whole of the cell (see Fig. 2, Thorn et al. 1993a). Low concentrations of ACh, which produce trains of short-lasting current spikes (Fig. 1B), induced transient elevations in [Ca²⁺]_i only in the secretory pole and coincident with each current spike (Thorn et al. 1993a).

Spatially restricted rises in [Ca²⁺]_i have been reported in oocytes (DeLisle and Welsh, 1992; Lechleiter and Clapham, 1992) in response to injection of InsP₃ and nonmetabolizable analogues of InsP₃. These usually gave rise to propagated [Ca²⁺]_i waves that spread away from the site of initiation, the foci of [Ca²⁺]_i elevations themselves have not been studied. There is also a report, in neurones, of [Ca²⁺]_i oscillations restricted to the distal ends of dendrites (Inagaki et al. 1991). No functional significance has been ascribed to either of these examples of spatially restricted spiking. We propose that the spatially restricted [Ca²⁺]_i spikes we describe form an important part of the functional secretory processes in pancreatic acinar cells.

A key advantage of the whole-cell patch-clamp technique is the access gained to the cell cytoplasm through the ability to perfuse the cell internally. One crucial series of experiments that has advanced our knowledge of oscillatory mechanisms showed that repetitive oscillations could be evoked by a constant level of InsP₃ (Wakui et al. 1989). In these experiments, a nonmetabolizable analogue of InsP₃ was infused through the patch pipette to provide a steady concentration of InsP₃ that could not be hydrolysed. These observations provide evidence against models of oscillations that require fluctuating concentrations of InsP₃ (Meyer and Streyer, 1988).

Other important experiments in the pancreas have shown that the infusion of heparin, a competitive antagonist at the InsP₃ receptor, can abolish the Ca²⁺ response to acetylcholine, InsP₃ (Wakui et al. 1990) and CCK (Thorn and Petersen, 1993b). These experiments illustrate the importance of this receptor in the oscillatory mechanism.

The agonist responses described in the previous section are thought to be due to [Ca²⁺]_i release through the action of InsP₃. Combined whole-cell patch-clamp and [Ca²⁺]_i imaging experiments have enabled us to test the sensitivity of different regions of the acinar cell to the infusion of InsP₃ (Thorn et al. 1993a).

In a series of experiments, Hassoni and Gray (1992) recorded the combined Ca²⁺-dependent currents and [Ca²⁺]_i signal in rat parotid acinar cells and showed the typical agonist-induced response to consist of an initial Cl⁻current associated with a luminal [Ca²⁺]_i elevation followed by the activation of a K⁺ current. In separate experiments, they then infused ‘caged’ InsP₃ through the patch pipette and recorded the Ca²⁺-dependent current response to the flash release of InsP₃ in rat parotid cells. Their results demonstrated current activation with a time course similar to that induced by agonist, suggesting a luminal-to-basal Ca²⁺ wave induced by this rapid release of InsP₃.

In our experiments on the pancreatic acinar cells, application of low concentrations of InsP₃ and other analogues elicited trains of current spikes (Wakui et al. 1989) and, after waiting for some minutes following breakthrough to whole cell, to allow for equilibration of the InsP₃, we then simultaneously recorded these Ca²⁺-dependent currents and the [Ca²⁺]_i response of the cell. An example of the results from an experiment infusing a nonmetabolizable analogue of InsP₃, InsP₃S₃, into a cell is shown in Fig. 3. Trains of Ca²⁺-dependent current spikes are seen in Fig. 3 (top traces); also shown are mean [Ca²⁺]_i changes measured from the basal pole of the cell and the secretory pole. The peak of each short-lasting Ca²⁺-dependent current spike is associated with a rise in [Ca²⁺]_i in the secretory pole of the cell with a return to basal levels of [Ca²⁺]_i between each of the spikes. The lower part of Fig. 3 shows three-dimensional plots of [Ca²⁺]_i obtained at the three time points shown and demonstrates again the spatially restricted nature of each [Ca²⁺]_i spike. In these experiments, we would expect the InsP₃ concentration to have equilibriated across the whole of the cell. The observation that only the secretory pole region responds to this level of InsP₃ provides direct evidence that this region of the cell has a higher sensitivity to InsP₃. In further experiments, we have shown that under certain conditions InsP₃ infusion can also reproduce the slow waves that spread across the cell (Thorn et al. 1992 a). The results we have obtained can explain the regional initiation of the [Ca²⁺]_i signal at high agonist concentrations and the repetitive spatially restricted Ca²⁺ spikes observed at lower agonist concentrations.

Further evidence of the high sensitivity to InsP₃ in the secretory pole was obtained from experiments in which we perfused ACh focally through a patch pipette after the formation of a cell-attached patch on the basal pole of the cell (Thorn et al. 1993a). Fig. 4 shows an example of one experiment: the elicited Ca²⁺ rise indicates that ACh receptors are located in the basal plasma membrane. The first Ca²⁺ response of the cell after ACh application showed a [Ca²⁺]_i rise simultaneously in both the secretory pole and the basal pole of the cell (Fig. 4 middle). It would be expected that, at this time, at the very initiation of the Ca²⁺ response, the InsP₃ concentration would have formed a gradient across the cell, high in the region immediately beneath the patch pipette and low in the secretory pole. The similarity in the time course and amplitude of the Ca²⁺ response in the two poles of the cell therefore provides further evidence that the Ca²⁺ store in the secretory pole has a higher sensitivity to InsP₃.

What is the basis of the high sensitivity of the secretory pole region to InsP₃? We have addressed this issue by studying the CCK-induced response, which consists of both the short-lasting local spikes and the broad transients. Infusion of a high concentration of the competitive InsP₃ receptor antagonist heparin (>250mgml⁻¹) (Ghosh et al. 1988) totally abolished the response (Thorn and Petersen, 1993b). Lower concentrations of heparin (<250 μg ml⁻¹) abolished only the local spikes, leaving the broad transients essentially unaffected. There are a number of problems associated with this use of heparin (Berridge, 1993; Thorn et al. 1993b), but one interpretation is that heparin is binding specifically to an InsP₃ receptor in the secretory pole that displays a higher heparin affinity. The affinity of the InsP₃ receptor for heparin has been correlated with its affinity for InsP₃ (Khan et al. 1992). A clustering of high-affinity InsP₃ binding sites in the secretory pole is consistent with the idea that a single cell may possess InsP₃ receptors of different affinity (Sharp et al. 1992).

In conclusion, Ca²⁺ imaging studies have provided evidence for initiation of the [Ca²⁺]_i signal in the secretory pole of the cell. Our studies using combined whole-cell patch-clamp and Ca²⁺ imaging have revealed local cytosolic spikes confined to the secretory pole region. InsP₃ infusion through the patch pipette also elicits [Ca²⁺]_i spikes restricted to the secretory pole and provides evidence for a region of high sensitivity to InsP₃ which may be explained by the regional localization of high-affinity InsP₃ receptors.

Ca²⁺ influx is not directly important in the mechanism of Ca²⁺ oscillations, which will persist for some time in the absence of extracellular Ca²⁺. We do know that, during each oscillation, a component of the Ca²⁺ released into the cytoplasm is actively extruded from the cells (Tepikin et al. 1992). The Ca²⁺ lost from the cell through extrusion eventually leads to the abolition of the oscillations during incubating in Ca²⁺-free extracellular media (Yule et al. 1991; Petersen et al. 1991a). In the presence of a normal extracellular Ca²⁺ concentration, therefore, the Ca²⁺ extruded must be balanced by an influx of Ca²⁺. In the pancreatic acinar cells, the Ca²⁺ influx pathway is not known but in the lacrimal acinar cells evidence has accumulated for a role for inositol tetrakisphosphate (InsP₄) in the activation of Ca²⁺ entry. It was originally shown that InsP₄ was able to facilitate Ca²⁺ entry in the presence of InsP₃ (Morris et al. 1987). Subsequent work demonstrated that a very high concentration of InsP₃ alone, infused into lacrimal cells, could evoke a Ca²⁺ influx (Bird et al. 1991). However, even at this very high concentration of InsP₃, there is still a demonstrable further potentiation of Ca²⁺ influx in response to the addition of InsP₄ (Smith, 1992). Much work remains to be done to clarify the nature and regulation of Ca²⁺ influx into exocrine acinar cells.

Ca²⁺ oscillations in acinar cells have provided key information contributing to our understanding of the mechanisms of Ca²⁺ handling in cells. Recent work has shown that regional differences in the Ca²⁺ signal exist within the acinar cells and has identified the secretory pole as an area of high sensitivity to InsP₃. The local spikes that we observe in this region in response to low agonist concentrations may provide a cellular mechanism for restricting the spread of the Ca²⁺ signal and therefore limiting the cell response to the elevation in [Ca²⁺]. The crucial role of InsP₃ receptors in the agonist-induced response is clear, but the mechanisms of the oscillations and the possible role of other types of Ca²⁺-releasing receptors remain unknown. We do not know whether oscillations are present in vivo and it could be that, at room temperature (used in the majority of studies), the normal processes of Ca²⁺ regulation are slowed so as to reveal oscillatory responses (Gray, 1988). It can be said that the activation of the Ca²⁺-dependent currents would occur physiologically and that these currents are crucial to all models of fluid secretion.

Kasai and Augustine (1990) proposed a model that depends on fluctuating membrane potential to provide a unidirectional flow of Cl⁻through the cell. In this model, the cell at rest is more hyperpolarized than the Cl⁻equilibrium potential. Agonist-induced oscillations evoke an initial opening of Cl⁻channels in the luminal plasma membrane, promoting Cl⁻efflux into the lumen. These channels then close, and nonselective channels and Cl⁻channels open in the basal plasma membrane. The nonselective channel depolarizes the cell to a value more positive than the Cl⁻equilibrium potential and Cl⁻flows into the cell through the Cl⁻channel on the basal membrane. The whole process is repeated at each oscillation. This model for fluid secretion is restricted to pancreatic acinar cells, which do not possess a Ca²⁺-dependent K⁺ channel. In other acinar cell types, this latter channel is thought to hold the cells hyperpolarized and to provide the outward driving force for Cl⁻exit to the lumen (Petersen and Gallacher, 1988). Against the Kasai and Augustine model, secretion still occurs during sustained elevations of [Ca²⁺] and does not therefore require the Ca²⁺ oscillations that are necessary in the ‘push–pull’ hypothesis. In models of secretion, the Cl⁻channels play a role in Cl⁻efflux, but active ion transport must be crucially important to provide the electrochemical gradients for fluid secretion to take place. Further work is necessary to build up a good model of fluid secretion for the rodent pancreas.

The high sensitivity to InsP₃ of the Ca²⁺ stores in the secretory pole region would be maintained in vivo and the repetitive local restricted Ca²⁺ spikes that we observe as a consequence of this sensitivity have a number of important implications. Phasic exocytotic secretion has been observed during Ca²⁺ oscillations in acinar cells (Maruyama, 1988) and also in gonadotrophs (Tse et al. 1993), with each global [Ca²⁺] rise being correlated with a burst of exocytosis. It is also known that exocytosis in acinar cells can be elicited when Ca²⁺ concentration is elevated in the secretory pole (Maruyama et al. 1993). Therefore, it would seem probable that the restricted Ca²⁺ spikes would elicit exocytotic secretion of digestive enzymes. The effective localization of the spikes in the secretory pole would limit the cellular consequence of Ca²⁺ stimulation and prevent activation of Ca²⁺-dependent processes, some of which are known to be damaging, in other parts of the cell. There are also energetic advantages for the cell in utilizing a local Ca²⁺ signal. The extrusion of Ca²⁺ after each global oscillation is a process that is energetically expensive (Tepikin et al. 1992); such costs would be reduced in the recovery from local spikes.

It has been proposed that oscillations may provide a means of digitally encoding an analogue stimulus (Berridge, 1993). It appears that in the pancreas there may be some elements of this, with an increased frequency of oscillations at increasing InsP₃ concentrations (Petersen et al. 1991b). However, it is also evident from the different patterns of oscillation that the spatial characteristics of the Ca²⁺ response are also very important.

It is known that there are a number of Ca²⁺ -dependent processes involved in exocytosis with different temporal characteristics (Neher and Zucker, 1993; Thomas et al. 1993). In response to a step change in [Ca²⁺]_i, three kinetically distinct phases of secretion were observed in rat pituitary cells (Thomas et al. 1993). An initial rapid phase of exocytotic release occurred within 40ms, followed by two slower phases, with the bulk of secretion occurring over 400–1000ms and a steady slow secretion for up to 8s. These time courses have been explained in terms of the relative position of the secretory vesicles to the plasma membrane. The initial release involves vesicles closely associated with the plasma membrane, followed by two subsets of vesicles positioned farther away (Thomas et al. 1993). In addition to the relative vesicle position, the process of regulated exocytosis is thought to involve changes in the cell cytoskeleton, with disassembly of actin filaments in a Ca²⁺-dependent process (Cheek and Burgoyne, 1991). This disassembly then allows vesicle access to the plasma membrane. Estimates of the time course of changes in the actin filaments (Cheek and Burgoyne, 1986) indicate that they are slow compared with the rapid exocytotic release described by Thomas et al. (1993). Initial cytoskeletal changes were observed after 5s and maximal changes after 30s, which shows some overlap with the slowest time course observed in the study of Thomas et al. (1993).

Actin filaments have been shown in the pancreatic acinar cells to be concentrated in the secretory pole (Drenckhahn and Mannherz, 1983) and cytoskeletal changes have been observed in parotid acinar cells during active secretion (Perrin et al. 1992). Comparison of the patterns of the Ca²⁺ oscillations evoked by CCK in the pancreas with the observed time courses of secretion allows some speculation as to the functioning of the Ca²⁺ signal. The short-lasting spikes in the pancreas were, on average, 2s in duration, which is close to the middle time course that elicits maximal exocytotic secretion described by Thomas et al. (1993). The broad transient oscillations that we observe have a width of approximately 30s. It is possible that these broad transient [Ca²⁺] rises are involved in priming the exocytotic process through a mechanism of cytoskeletal change and movement of vesicles closer to the plasma membrane (Fig. 5). Such a mechanism would be able to regulate secretion over long periods. If secretion took place by a simple [Ca²⁺] rise, the rate of secretion would be limited by the kinetics of the exocytotic events. However, in mechanisms of periodic exocytosis, the rate of secretion would be determined precisely by the frequency of oscillations.

The evidence from our laboratory (Thorn et al. 1993a) indicates that infusion of InsP₃ can simulate the agonist response in acinar cells. Unfortunately, heparin and decavanadate, both used as antagonists at the InsP₃ receptor, have rather nonspecific actions and better tools are required to push our knowledge further. It has already been shown that monoclonal antibodies can be used against the InsP₃ receptor to block propagated waves in oocytes (Miyazaki et al. 1992). In the future, antibodies may be raised against specific subtypes of the InsP₃ receptor (Ross et al. 1992) to test the hypotheses we have put forward about receptor distribution.

In addition to the InsP₃ receptor, there is evidence in acinar cells for ryanodine effects (Foskett and Wong, 1991; Nathanson et al. 1992) and Ca²⁺ effects (Wakui et al. 1990) and there is also evidence from single-channel studies of reconstituted internal membranes (Schmid et al. 1990) that indicate a diversity of other Ca²⁺-releasing receptors. The role of these latter receptors remains unknown and may involve ‘sensitizing agents’, such as cyclic ADP ribose, in their activation (Galione, 1993). One example of Ca²⁺ mobilization from another unknown Ca²⁺ pool comes from experiments on a CCK analogue, JMV-180, which induces a Ca²⁺ release in permeabilized cells that is apparently independent of caffeine-or InsP₃-sensitive stores (Saluja et al. 1992). In our own studies, we have shown that JMV-180 elicits oscillations of mixed pattern like those induced by CCK and that these can be blocked by heparin (Thorn and Petersen, 1993c). Once again, this highlights the role of the InsP₃ receptor in oscillations. However, it does not address the issue of a separate Ca²⁺ pool that could release Ca²⁺ and subsequently sensitize the InsP₃ receptors (Bezprozvanny et al. 1991). Sensitization of InsP₃ receptors by sulphydryl reagents has recently gained some significance (Missien et al. 1991; Thorn et al. 1992; Bootman et al. 1992), and we have proposed that a similar sensitizing action may be involved in the agonist-induced response (Thorn et al. 1993a).

In summary, recent experiments have revealed a complex temporal and spatial pattern of Ca²⁺ signalling. Evidence is accumulating that intracellular variation in sensitivity to InsP₃ can play a role in the production of different Ca²⁺ signals. This complexity would allow the cell a repertoire of different physiological responses. The challenge for future investigation will be to determine the links between the Ca²⁺ signal and the physiological processes.

I would like to thank Professor Ole Petersen for continuing help and guidance throughout my years at Liverpool. I would also like to thank Professor R. D. Burgoyne for discussion and advice on this manuscript.