Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells

David R. Giovannucci, David I. Yule, and Edward L. Stuenkel

Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance (Cm) measurements and by the membrane fluorescent dye FM1-43. When measured with the Cm method, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3.5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s-1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of Cm changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.

exocytosis; endocytosis; FM1-43; membrane capacitance; fura 2

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

STIMULATION OF PANCREATIC acinar cells with cholecystokinin (CCK) or ACh evokes the release of digestive enzymes by activating a heterotrimeric GTP-binding protein-linked, phospholipase C-mediated, signal transduction cascade that rapidly elevates the cytosolic Ca2+ concentration ([Ca2+]i) (25, 47). Within the acinar cell, digestive enzymes are packaged into a relatively homogenous population of zymogen granules (ZG) with a diameter of 0.83 µm (2) and released into the lumen of the pancreatic ducts via exocytotic fusion at the cell's apical region. After exocytotic activity, the ZG membrane is retrieved from the plasma membrane by a process that may involve clathrin and dynamin (15, 21, 41, 43). Despite significant advances in the understanding of receptor activation, signal transduction, and Ca2+-signaling pathways in acinar cells, detailed information on the molecular machine controlling the exocytotic release of digestive enzymes or the retrieval of membrane after secretory activity is lacking. Insights into the most rapid of these events can be provided by the use of methods that allow the monitoring of membrane turnover with high temporal and spatial resolution at the level of the single cell. For example, in isolated nerve endings and in single neuroendocrine cells, temporally resolved cell membrane capacitance (Cm) measurements, made by the whole cell patch-clamp method, have been widely used to track exocytotic activity with millisecond time resolution. This method has revealed functionally distinct vesicle pools that give rise to multiple secretory phases (8, 11, 12, 26) and has been used to probe the functional interaction of proteins involved in vesicle docking and fusion (5, 13, 15, 16). Furthermore, the use of Cm measurements has revealed multiple forms of endocytosis after exocytotic activity that differ in their kinetics and Ca2+ dependence (1, 6, 7, 39).

The Cm method has recently been applied to study the exocytotic activity of exocrine cells (13, 22, 23, 28, 31). However, the close spatial and temporal coupling of exocytotic and endocytotic processes suggest that the Cm method alone, despite unparalleled temporal resolution, may not be sufficient to independently resolve these processes. This concern is of specific importance when the Cm method is used to investigate exocytosis and endocytosis in pancreatic acinar cells, which secrete at a slower rate and for longer time periods than do neurons. Recently, optical measurements of membrane turnover have been developed that use membrane-sensitive fluorescent probes that can provide real-time measurements of secretory dynamics (19, 24, 28, 33, 35). These methods allow for the independent evaluation of exocytotic and endocytotic activity and report spatial information regarding these processes. The most widely used of these probes is FM1-43, a fluorescent amphipathic styryl dye that rapidly and reversibly partitions into the outer leaflet of biological membranes, where its quantum yield increases ~300-fold (35). For example, FM1-43 used in combination with confocal or fluorescence deconvolution microscopy has been used to track secretion at the single cell level (19, 33, 35) and to monitor the cycling of single 50-nm synaptic vesicles (30). Because FM1-43 is nearly nonfluorescent in aqueous solutions, it provides an excellent signal-to-noise ratio, even under conditions of limited membrane cycling. Moreover, FM1-43 is a convenient endosomal marker, since it cannot penetrate the membrane completely and becomes trapped in endocytosed vesicles. Thus this on-line optical measure of cumulative exocytotic activity can provide spatial information regarding the sites of exocytotic/endocytotic activity and the fate of endocytosed membrane. It was a specific goal of the present study to apply FM1-43, as well as Cm measurements, to probe the secretory activity of pancreatic acinar cells and provide spatial and temporal information on resting and agonist-evoked exocytotic and endocytotic activity at the single cell level. The data indicate that exocytotic and endocytotic activity are nearly coincident and demonstrate the feasibility of an optical method to monitor exocrine cell secretion. In addition, membrane retrieval after exocytosis was shown to be confined to the cells' apical regions.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of acutely isolated rat pancreatic acinar cells. Acini and single acinar cells were prepared from pancreata of male Sprague-Dawley rats (200-250 g) according to a collagenase digestion procedure (45, 46). Isolated acini were incubated at 37°C in a physiological salt solution (PSS) containing (in mM) 145 NaCl, 2.5 K2HPO4, 1.0 CaCl2, 1.0 MgSO4, 10 glucose, and 10 HEPES, 0.5 mg/ml BSA, and 0.1 mg/ml soybean trypsin inhibitor; pH was adjusted to 7.4 with NaOH. For FM1-43, fura 2, and patch-clamp experiments, acini were aliquoted onto clean glass coverslips placed in perifusion chambers. All the acinar cells in this study were used within 3 h of isolation, and only those acinar cells that exhibited well-defined polarity were studied.

Cm measurements. The use of voltage-clamped single acinar cells under the whole cell patch-clamp configuration allowed for the measurement of small, time-resolvable changes in Cm using a modified phase-tracking method, as previously described (8). Briefly, the Delta Cm of single acinar cells was measured by monitoring, at two orthogonal phase angles, the current response to a 30-mV root mean square sine wave at 1,201 Hz applied over a holding potential of 0 or -30 mV. The correct phase angle was determined using a software-based phase-sensitive detector (Pulse Control software, courtesy of Dr. Richard Bookman, University of Miami, Miami, FL). Calibration pulses of 100 fF and 500 kOmega were generated at the beginning of each trace, and Cm was determined at 6.6-ms intervals. Patch pipettes were filled with a solution that contained (in mM) 140 N-methyl-D-glucamine (NMDG) chloride, 40 HEPES, 2 ATP (Mg2+ salt), 0.2 GTP, and 0.25 Tris-EGTA; pH was adjusted to 7.1 with Tris. For experiments in which the level of cytosolic free Ca2+ was set at 31.4 µM, the pipette solution contained (in mM) 105 NMDG-glutamate, 48 NMDG-HEPES, 2 HEDTA, 1.5 Ca(OH)2, 2 ATP (Mg2+ salt), and 0.2 GTP; pH was adjusted to 7.0 with Tris. Series resistance averaged 10.8 ± 1.8 MOmega .

Fluorescence measurements and confocal microscopy. Experiments using the membrane probe FM1-43 as an on-line assay of secretory activity in acini were conducted using xenon (450-W) or mercury (100-W) lamp epifluorescence/photon-counting methods (8) and confocal microscopy (Noran/Oz system, Middleton, WI). For confocal experiments, FM1-43 was excited by the 488-nm laser light line from the krypton-argon laser, and emitted light passed through a 500-nm light long-pass filter before measurement of fluorescence intensity. Because prolonged exposure to the laser light was phototoxic to acinar cells, laser light was set at 10% power and applied for only 33 ms at intervals to take a snapshot image. The optical thickness was typically 1 µm and taken through an equatorial region of an acinus. Image analysis was performed using Intervision software on a Silicon Graphics INDY workstation.

For secretion-coupled membrane turnover and [Ca2+]i dynamics, 4 µM FM1-43 or 1 µM fura 2-AM (Molecular Probes, Eugene, OR) was applied by local or bath perifusion in PSS. Individual acinar cells were optically isolated using a 20-µm pinhole stop and then illuminated by epifluorescence through a ×40 oil immersion objective (1.30 NA). In those cases where simultaneous measurement of FM1-43 and fura 2 fluorescence was required, alternating excitation wavelengths of 488 and 380 nm were used. The fluorescent intensities at an emission wavelength of 530 nm, corresponding to each excitation light, were measured by a photomultiplier.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Membrane turnover monitored by Cm measurements. Figure 1A shows a representative, agonist-evoked change in Cm in an isolated acinar cell measured using whole cell voltage clamp. Acinar cells responded to Ca2+-mobilizing agonist application (10 µM carbachol) with an average increase in cell Cm of 268 ± 62 (SE) fF (n = 6) over a time period of several seconds. The average resting Cm of these cells was 6.8 ± 1.5 pF, indicating that this secretory response transiently increased the cell surface area by 3.5%. After removal of carbachol, the Cm signal rapidly returned to prestimulus values. In two cells, however, recovery of the resting Cm was only partial after agonist removal. On the basis of a specific Cm of 10 fF/µm2, the total secretory response could be accounted for by 12 ZG fusions at a rate of 1-2 s-1, with the assumption that each fusion event contributes an additional 2.2 µm2 of membrane to the surface (see DISCUSSION). These calculations assume that there was no concurrent endocytotic activity. In most cases, these stimulus-induced increases in Cm were accompanied by substantial decreases in membrane resistance and increases in [Ca2+]i. In three cells (not included in our analysis), a decrease in Cm, likely representing excess endocytotic activity, was observed in response to 10 µM carbachol application.


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Fig. 1.   Agonist-evoked changes in surface area of isolated acinar cells estimated by temporally resolved membrane capacitance (Cm) measurements. A: application of 10 µM carbachol (CCh) evoked a transient increase in acinar cell surface area. B: effect of application of 10 µM CCh to an acinar cell internally dialyzed with 100 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). Agonist treatment evoked stepwise increase in Cm (tick marks). C: amplitude histogram of step Cm increases from 2 cells treated with both GTPgamma S and CCh. D: averaged amplitudes of Cm increase of single acinar cells evoked by treatment with CCh, GTPgamma S, or CCh and GTPgamma S.

A number of features of the Cm data suggested a temporal coincidence of exocytotic and endocytotic activity in acinar cells: 1) the relatively slow, modest evoked changes in Cm, 2) the failure to resolve single ZG fusion events, and 3) the variability of endocytotic activity. To specifically test the hypothesis that endocytotic activity occurs in combination with the exocytotic rise in Cm in acinar cells, we attempted to arrest the membrane retrieval process by including 100 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) in the patch pipette solution (1). This experimental rationale was based on a preponderance of evidence implicating the GTPase dynamin in the internalization of membrane via clathrin-coated vesicles (18, 21, 41, 43) or a clathrin-independent mechanism (1). As shown in Fig. 1B, when secretion was evoked in an isolated acinar cell by agonist application, in combination with GTPgamma S dialysis, a robust, often stepwise increase in Cm was induced. On average, this Cm increase was 1,120 ± 346 fF (representing the fusion of ~51 ZGs), and whole cell Cm was maintained at a level corresponding to this exocytotic increase (n = 2). The average size of the largest observable steplike events was 38.6 ± 4.9 fF (n = 23). As shown in Fig. 2C, however, the frequency of these step events was distributed between two sizes averaging 19.7 and 41.6 fF, corresponding to the fusion of single granules with diameters of 0.79 and 1.15 µm, respectively. Although these size estimates are within the range of reported ZG diameters (2, 22), the possibility of fusion of compound vesicles cannot be excluded. We observed, however, that dialysis of GTPgamma S via the patch pipette also activated exocytotic release and evoked a slow, modest rise in Cm in the absence of a Ca2+-mobilizing stimulus, with stepwise increases in Cm and no endocytotic recovery of the baseline Cm. As shown in Fig. 1D, this increase was 218 ± 78 fF on average (n = 3). As such, a possible synergistic action of Ca2+ and GTPgamma S to increase amylase release cannot be excluded.


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Fig. 2.   Fluorescence measurements of single acinar cell membrane dynamics. A: surface-accessible membrane measured by photometry using 4 µM FM1-43 while in cell-attached patch configuration. Conversion to whole cell configuration was performed at arrowhead with a solution buffered at 31.4 µM free Ca2+. B and C: monitoring of FM1-43 fluorescence as in A, with a pipette solution containing 10 µM EGTA and no added Ca2+ (B) or Ca2+ and GTPgamma S (C). D: effect of application of 1 µM CCh (at arrowhead) in presence of FM1-43 on intact acinar cell fluorescence (c and b). Cell fluorescence after washout of secretagogue and dye returned to a resting level above prestimulus value (a and d), reflecting FM1-43 accumulation by endocytosis. E: comparison of averaged FM1-43 fluorescence increases evoked in single acinar cells after equilibration of cytosol with 31.4 µM Ca2+ (+Ca) or 10 mM EGTA or 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (-Ca), Ca2+ in combination with 100 µM GTPgamma S, or 1-10 µM CCh.

Membrane turnover monitored by FM1-43 fluorescence. The membrane dye FM1-43 (a probe of cumulative exocytotic activity) provided an alternative method for monitoring secretion-coupled membrane turnover in single pancreatic acinar cells. As shown in Fig. 2A, local application of 4 µM FM1-43 in PSS to a single cell or small cluster (doublet or triplet) of acinar cells caused a rapid, membrane-delimited increase in fluorescence. This fluorescence reached a plateau value [time constant (tau ) = 42 ± 8 s, n = 7], where in the absence of secretagogue it remained constant or increased slowly over minutes. On removal of FM1-43 from the bath, plasma membranes were rapidly destained and fluorescence fell to a level equal to or only slightly higher (<5%) than preload levels.

In initial experiments, attainment of the whole cell patch-clamp configuration was used to clamp [Ca2+]i at 31.4 µM to evoke a secretory response in single acinar cells (Fig. 2A). This [Ca2+]i has been shown to be half-maximal at eliciting secretion in permeabilized acini (27). After rupture of the patch membrane, the FM1-43 fluorescence signal increased, on average, by an additional 22.6 ± 4.5% (Fig. 2E) and rose to this new plateau value with a tau  of 162 ± 65 s (n = 3). Removal of the FM1-43 from the bath solution and from the surface membrane reduced the acinar cell fluorescence to a steady-state level higher than that measured before the attainment of whole cell configuration. This new level was roughly equal to the evoked fluorescence increase and indicated that the FM1-43 dye was rapidly internalized. On the other hand, only a modest, gradual fluorescence increase was evoked in three of four cells after attainment of whole cell configuration using a pipette solution containing 10 mM EGTA or 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid with no added Ca2+ (Fig. 2B). The average increase in fluorescence in these cells was 4.8 ± 3.1% (n = 4) and was significantly smaller than that evoked in cells dialyzed with Ca2+. The data indicate that the majority of the increase in acinar cell fluorescence represented Ca2+-dependent exocytotic/endocytotic activity. Next, we demonstrated that whole cell dialysis with 31.4 µM Ca2+ in combination with 100 µM GTPgamma S could evoke an increase in the amount of labeled membrane but significantly reduce its subsequent internalization (Fig. 2C), as indicated by a return of fluorescence to prestimulus levels after washout of the dye. As shown in Fig. 2E, the average fluorescence increase was 28.8 ± 12.9% (n = 4). Removal of FM1-43 rapidly reduced the cell fluorescence to a residual level equal to only 10.5 ± 5.3% of the evoked increase. This indicated that most of the FM1-43 dye remained surface accessible, consistent with the hypothesis that GTPgamma S inhibits endocytotic fission.

To determine whether a similar increase in fluorescence could be elicited in intact acinar cells (without the use of the whole cell patch-clamp method), we treated single cells or small clusters of cells with 1-10 µM carbachol in the continuous presence of FM1-43 (Fig. 2D). Introduction of a secretory stimulus was shown to evoke an additional fluorescence increase in 7 of 13 cells. Figure 2E shows that, during continuous application of 10 µM carbachol, acinar cell fluorescence increased by an additional 13 ± 1.7% (n = 7). The increases usually consisted of a rapid initial rise with a tau  of 223 ± 63 s, followed by a slower increase in fluorescence. These data are consistent with exocytotic fusion inducing a fluorescence increase as additional membrane is exposed to the dye. Because the fluorescent properties of the dye in endosomes are essentially the same as in the plasma membrane, FM1-43 is also a specific marker of activity-dependent membrane reuptake (33, 35). As such, after removal of the dye from the perifusion medium and destaining of the plasma membranes, acinar cell fluorescence returned to a level higher than preload value. This new steady-state value was, on average, 65 ± 16% of the stimulus-evoked rise in fluorescence (n = 7). This increase was interpreted to reflect the endocytosis of dye-labeled membrane.

A correlation between the fluorescence increase and a rise in [Ca2+]i was demonstrated in a set of experiments in which intact acinar cells were loaded with the Ca2+-sensitive dye fura 2-AM before loading with FM1-43. Consistent with previous studies (19, 33), there was no optical cross-talk between changes in FM1-43 and fura 2 fluorescence in cells loaded with both dyes. Excitation at 380 nm (fura 2) and 488 nm (FM1-43) was used to simultaneously monitor the rise in [Ca2+]i and the increase in FM1-43 fluorescence in response to agonist application (n = 3). As shown in Fig. 3A, 1 nM CCK evoked a transient increase in [Ca2+]i and a sustained rise in FM1-43 fluorescence. In addition, using optical image analysis, we found that CCK treatment was sufficient to evoke an increase in FM1-43 fluorescence in whole acini. For these experiments, we selected small, isolated acini (usually ~6-12 cells), preferably of one cell layer thickness. Whole acini were observed to stain with FM1-43 with tau  of 165 ± 14 s (n = 6). As with isolated acinar cells, a rise in FM1-43 fluorescence in response to agonist application was not always observed. However, in those acini that clearly exhibited rises in FM1-43 fluorescence, agonist application evoked a 29 ± 8.5% fluorescence increase with an average tau  of 366 ± 69 s (n = 5). Figure 3B shows an example of the total evoked fluorescence change in an acinus composed of six to eight cells. On average, the percent increases were larger than those determined for isolated acinar cells.


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Fig. 3.   Correlation of membrane turnover with changes in cytosolic Ca2+. A: effect of application of 1 nM cholecystokinin (CCK). Changes in cytosolic Ca2+ were monitored by fura 2 (excited at 380 nm); increase in surface-accessible membrane was simultaneously monitored by FM1-43 fluorescence (excited at 488 nm). Dashed line, steady-state cell fluorescence after loading. B: effect of agonist stimulation with 1 nM CCK on staining of surface-accessible membranes of an intact acinus composed of 8 cells loaded with 10 µM FM1-43 at 37°C. CCK treatment evoked a significant increase in acinus fluorescence, which, after removal of dye from perfusate, decreased to a new steady-state level greater than prestimulus value.

Dye accumulation and subcellular localization after exocytotic activity. In acinar cells the receptor binding and signal transduction machinery are localized to the cell's basolateral surface, whereas the ZGs and the sites of Ca2+ release, or the "trigger zone," and exocytotic fusion are confined to the apical region. This suggests that the sites of ZG exocytosis are close to the sites of Ca2+ release. This high degree of morphological and functional partitioning suggested that it might be possible to localize sites of poststimulus FM1-43 fluorescence. We tested the hypothesis that exocytosis and endocytosis are not only functionally linked but colocalize as well. For these experiments, groups of acinar cells were loaded with 10 µM FM1-43 in a closed perifusion chamber mounted on a confocal microscope. Secretion was then evoked by treatment with 10 µM carbachol or 1 nM CCK in the continuous presence of dye. After stimulation the cell was washed free of FM1-43, enabling localization of the endocytosed dye. Confocal images were acquired before stimulation and immediately after the dye was washed off to image the sites of membrane retrieval.

Figure 4 shows a bright-field and corresponding confocal fluorescence images of an acinar cell cluster. Addition of FM1-43 to the bath solution rapidly stained the plasma membrane but did not label internal membranes. No further increase in fluorescence was observed in the absence of agonist, suggesting that acinar cells have relatively low resting levels of membrane turnover. However, over 5 min in the continued presence of FM1-43 and 1 nM CCK, the cytosolic signal was gradually increased. After 12 min the dye was removed from the bath solution and the plasma membrane was rapidly destained, leaving apically delimited punctate staining. In addition, patterns of basolateral staining as well as diffuse cytoplasmic staining that did not correspond to any known organelles were observed. The punctate pattern of staining presumably reflected internalization of postfusion surface membrane near the sites of exocytotic activity. This punctate staining was different from the diffuse perinuclear staining that was observed in other preparations where cells were exposed to laser light for longer periods and at higher power. Such cells contained large vacuoles and were obviously damaged by phototoxic events (data not shown). Measurement of cell sizes before and after stimulus indicated that the surface area of the acinar cells remained relatively constant, suggesting that exocytosis and endocytosis were roughly balanced.


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Fig. 4.   Bright-field (A) and corresponding confocal fluorescence images (B-D) of a rat pancreatic acinus. No fluorescence was detectable before application of FM1-43 (B). Addition of FM1-43 to bath rapidly labeled surface-accessible membranes (C). After treatment with 1 nM CCK, dye was removed from bath solution and plasma membrane rapidly destained, revealing punctate, apically delimited staining (D).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Time-resolved Cm measurements have the necessary temporal resolution and sensitivity for determining the initial kinetics and Ca2+ dependence of ZG fusion and have been applied to single acinar cells by a number of researchers (13, 23, 29, 31). In the present study the percent area increase evoked by Ca2+-mobilizing agonist application as estimated by the Cm method was ~3.5% of the resting whole cell Cm, at an estimated initial rate of one to two ZG fusions per second. The magnitude and time course of this evoked rise in Cm were consistent with those previously reported for rat single acinar cells (13, 23). With the assumption that most ZGs are spherical with a diameter (d) of 0.83 µm (2) and with a specific Cm of 10 fF/µm2, the evoked change in Cm can be readily converted to the number of fused ZGs. Thus, per fusion event, each ZG should add 22 fF to the whole cell Cm. However, the Cm increased in a smooth, gradual fashion, which is surprising because the Cm methods used in this study have the necessary resolution to detect single ZG fusions. This suggested that the Cm measurements may be complicated by the projection of conductance changes or by the contribution of rapidly activated endocytotic activity. Under our recording conditions, large changes in membrane conductance or series resistance almost always accompanied the evoked change in Cm. However, manual dithering of the whole cell capacitance during the agonist-evoked Cm changes did not induce corresponding changes in the conductance trace, indicating that the phase angle was properly adjusted (data not shown). Consistent with the latter interpretation, intracellular perfusion of GTPgamma S has been shown to block stimulus-evoked endocytotic activity in nerve endings (38) and chromaffin cells (1). Although a role for dynamin in the retrieval of ZG membrane after exocytotic activity in acinar cells is speculative, splice variants of the dynamin II isoform have been demonstrated in multiple cell types (6, 41, 42), and recently dynamin II was shown to associate with the Golgi and the plasma membrane in isolated hepatocytes (15). In the current study, when GTPgamma S was included in the pipette, the average evoked increase in acinar cell area was estimated to be 14.6%, and additionally a substantial increase was observed in the number of stepwise events. This increase corresponds to the fusion of ~51 ZGs. This value was similar to the amount of membrane turnover in response to carbachol treatment estimated by the increase in FM1-43 fluorescence. In those cells the average fluorescence increase was 13 ± 1.7%. The fourfold larger increases in surface area observed under conditions presumed to block endocytotic activity indicate that, in acinar cells, when secretory activity is assayed by the Cm method, the rate and magnitude of continuous exocytotic activity may be masked as membrane retrieval matches or overtakes the exocytotic rate.

Using FM1-43 to track membrane turnover allows for estimation of cumulative exocytotic activity. For example, given that the surface area (pi d2) of an isolated, spherical acinar cell with d = 16 µm is ~814 µm2 (see below), the number of ZGs fused can be quantified, with the assumption that 1) the FM1-43 fluorescence is not significantly altered by dye uptake into endosomes, 2) fusion-competent ZGs are of a relatively consistent size, and 3) ZGs undergoing exocytosis are fully loaded with dye. For example, on average, the loading of an acinar cell's plasma membrane with FM1-43 increased the relative fluorescent signal by 3.65 units. This increase corresponds to a specific fluorescence of 0.0045/µm2 membrane. Each ZG fusion should contribute an additional 2.2 µm2 of FM1-43-accessible membrane (dZG ~ 0.83 µm), corresponding to an additional increase of 0.27% to the relative fluorescence. Thus, on average, carbachol stimulation was estimated to evoke the exocytotic fusion of ~48 ZGs over a period of 90 s.

Comparison of our estimates of the magnitude and rate of the secretory response of single acinar cells with measurements of amylase release obtained from permeabilized cells requires an estimate of the number of ZGs per acinar cell. In a recent study, Aughsteen et al. (2) used stereological methods to demonstrate that in rat acinar cells ~8.3% of the cell volume is occupied by ZGs. In the present study the average diameter of the acinar cells was 16 ± 5 µm with a mean volume of 2,185 µm3 (n = 16). Thus we estimated that there were ~600 ZGs/cell. In permeabilized rat acinar cells, 30 µM Ca2+ has been reported to evoke the release of ~6 or 9% of the total amylase content after 2.5 and 5 min, respectively (27). This would correspond to the fusion of 36-54 ZGs at a rate of 0.2 s-1, similar to our FM1-43-based estimate, indicating the fusion of 48 ZGs at a rate of 0.5 s-1.

FM1-43-based estimates of the magnitude of secretory activity should be considered an upper limit, because, in addition to reporting the fusion of ZG membrane with surface membrane, FM1-43 may label additional membrane after hormonal activation of endocytotic activity and other membrane trafficking events. For example, endocytotic vesicles containing dye may label additional membrane via fusion with unlabeled internal membrane systems (33). Moreover, hormonal stimulation may contribute to membrane turnover by activating receptor trafficking events, the expression or downregulation of other plasmalemmal proteins, or the exposure of additional luminal membrane accessible to FM1-43 staining.

CCK-evoked increases in membrane turnover were also detected in whole small acini stained with FM1-43. On average, these percent increases were greater than those induced in single or small clusters. This observation is consistent with permeabilized cell studies, where amylase secretion from acini has been shown to be greater than that measured from isolated acinar cell suspensions (36, 45). Stripped of junctional contacts, enzymatically isolated acinar cells lose some of the structural features of apical specialization. For example, the loss of microtubules and apical microvilli and swelling of the luminal membrane have been reported (44). However, the molecular machinery necessary for signal transduction and amylase secretion remains largely intact. In the current study, in contrast to measurements of secretion from a bulk population of isolated cells, only single cells that retained their polarized morphology, with ZGs organized around the secretory pole, were selected for study. In these polarized acinar cells, many of the functional characteristics that ensure efficient amylase release are retained. For example, hormonal stimulation can still evoke a rise in Ca2+ that initiates at a distinct region in the luminal pole, and a differential distribution of Ca2+ extrusion sites between the luminal and basal poles of isolated cells has been reported (3, 40). After hormonal stimulation, removal of FM1-43 from the perfusate destained the surface membranes of acini, but the fluorescence did not return to prestimulus levels. This indicated that FM1-43 accumulated within acinar cells during the stimulus. Confocal microscopy revealed that much of the internalized dye was localized to small endosome-like structures, mostly confined to the apical regions of the acinar cells. In addition to this pattern of staining, there was a concomitant uptake of dye basolaterally, as well as diffuse cytosolic staining. It is conceivable that stimulation can activate membrane internalization that is not associated with exocytotic activity.

In neurons the rapid recovery of secretory vesicle membrane subsequent to the exocytotic fusion process is achieved by an endocytotic fission process, which has been estimated by capacitance and by optical methods to occur with half times of 1-20 s (37). This membrane retrieval is achieved via clathrin-coated vesicles and endosomes or uncoated invaginations after periods of intense activity or by the retrieval of vesicles that have undergone only partial fusion with the plasma membrane during conditions of moderate activity (37). The extent to which these processes might apply to the endocytotic mechanism of acinar cells, where ZGs presumably do not refill but rather recycle after fusion, is not known, although there appear to be some qualitative similarities. For example, when measured by patch-clamp methods, endocytosis in rat pancreatic cells was shown to require extracellular Ca2+ and may occur with a half time on the order of seconds (23). Also, recent work by Schneider et al. (32) using atomic force microscopy suggested that most amylase secretion occurs through pores that are formed by the transient fusion of the ZG membrane with the plasma membrane. This would suggest that acinar cells do not necessarily undergo surface expansion of the luminal pole and that granules do not collapse into the surface membrane. However, different patterns of endocytotic activity may correlate with the type or intensity of the secretory stimulus. In addition, consistent with the current study, a fluid phase marker (dextran) was shown to rapidly redistribute to the Golgi, vacuoles, and mature ZGs in acinar cells after stimulus-evoked secretion (9, 10, 20).

The use of an optical method for monitoring the membrane turnover of single acinar cells, in combination with patch-clamp-based measurements of cell surface area, has revealed that hormone-activated exocytotic and endocytotic activities arise within seconds and occur concurrently. Furthermore, the use of FM1-43 in combination with confocal microscopy demonstrates that exocytotic activity and membrane retrieval are functionally and spatially linked.

    ACKNOWLEDGEMENTS

We thank Drs. John A. Williams and Michael Hlubek for critical readings of the manuscript, Thomas Komorowski for help with the figures, and Dr. Glen Morangie for thoughtful insight. Confocal microscopy and image analysis were performed at the Michigan Diabetes Research and Training Center's morphology core facility.

    FOOTNOTES

This work was supported by National Institutes of Health Grants NS-36227 (to E. L. Stuenkel) and DK-41122 (to J. A. Williams) and grants from the Michigan Gastrointestinal Peptide Center (to D. R. Giovannucci and D. I. Yule).

Present address of D. I. Yule: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 711, Rochester, NY 14642.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. R. Giovannucci, Dept. of Physiology, University of Michigan Medical School, 7804 Medical Sciences II Bldg., Ann Arbor, MI 48109-0622.

Received 11 March 1998; accepted in final form 11 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(3):C732-C739
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