Correspondence to: Bertil Hille, Department of Physiology and Biophysics, G-424 Health Sciences Building, University of Washington, Seattle, Box 357290, WA 98195-7290. Fax:206-685-0619 E-mail:hille{at}u.washington.edu.
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Abstract |
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We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca2+ using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca2+ was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca2+, PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.
Key Words: secretion, secretagogue, cyclic AMP, photometry, amperometry
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INTRODUCTION |
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In neurons and some endocrine cells, Ca2+ plays a pivotal role as the final signal for rapid stimulus-evoked release of neurotransmitters and hormones. An elevation of intracellular Ca2+ will also trigger exocytosis in various nonexcitable cells (
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METHODS |
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Chemicals
Stock solutions of 20 mM forskolin, 20 mM H-89, 100 µM phorbol-12-myristate-13-acetate, and 500 µM bisindolylmaleimide I (BIS)1 were prepared in dimethyl sulfoxide. Stock solutions of 1 mM epinephrine, 1 mM vasoactive intestinal peptide (VIP), and 1 M cAMP analogues were made up in saline solution. The cAMP analogues included 8-Br-cAMP and the Rp and Sp optical enantiomers of 8-Br-cAMPS. Rp-8-Br-cAMPS, Sp-8-Br-cAMPS, H-89, PMA, and BIS were purchased from Calbiochem. VIP was from Bachem. Serotonin-HCl and dopamine-HCl were from RBI. Other chemicals were from Sigma-Aldrich.
Cell Culture
The derivation and culture of pancreatic epithelial cells from the main pancreatic duct of the dog has been described (
For single-cell experiments, cells were plated on small coverglass chips (5 x 5 mm) coated with a thick layer of Vitrogen. The cells were fed with medium conditioned by gallbladder myofibroblasts. Experiments were performed with cells between 2 and 7 d after plating. Although the epithelial cells we use can form well differentiated monolayers with apical-basolateral polarity, our experiments were carried out on subconfluent epithelial cells that were presumably not completely differentiated or polarized.
Loading of Monoamines and Amperometric Measurement of Exocytosis
We used carbon-fiber amperometry to detect exocytosis from single cells in real time (
For loading, cells were incubated for 40 min at room temperature in a solution containing (mM): 70 serotonin or dopamine, 68 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES, pH 7.3 with NaOH. The cells were then transferred to culture medium and kept at 37°C, 5% CO2 for up to 4 h. Experiments were performed in a monoamine-free saline solution containing (mM): 138 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES, pH 7.3 with NaOH.
Vesicular release of catecholamine was monitored as pulses of electric current generated by oxidation of the molecules at the tip of a carbon-fiber electrode polarized at +600 mV. Carbon-fiber microelectrodes were fabricated from 511-µm carbon fibers (PAN T650 or P25; Amoco Performance Products) and polypropylene 10 µl micropipettor tips as described by
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Solutions were exchanged by a local perfusion system that allows complete exchange of medium bathing the cells within 2 s.
Single-Cell Photometry
The intracellular Ca2+ concentration ([Ca2+]i) was measured with the Ca2+-sensitive fluorescent dye indo-1 loaded into cells such as the membrane-permeable AM ester (Molecular Probes, Inc.). The cells were incubated for 30 min in saline solution with 1 µM indo-1 AM at room temperature. The excitation wavelength was 365 nm (100 W mercury lamp), and fluorescence signals were recorded at 405 and 500 nm, using a pair of photon-counting photomultiplier tubes. The sample rate was 0.5 Hz for photometry alone and 0.1 Hz for simultaneous photometry and amperometry. Background fluorescence measured from a cell-free area was corrected. The [Ca2+]i was calculated using the equation: [Ca2+]i = K* (R - Rmin)/(Rmax - R), where R is the 405/500-nm fluorescence ratio and Rmin and Rmax are the ratios for Ca2+-free and -bound dye, respectively (
For measuring [Ca2+]i levels higher than several micromolar, some cells were loaded with the low-affinity dye, mag-indo-1. Loading of the dye was performed as for indo-1, except for a shorter loading time (510 min). The calibration solutions were the same, except the mid-point solution contained 10 mM nitrilotriacetic acid and 5 mM Ca2+ with a calculated free [Ca2+] of 100 µM. Values for Rmin, Rmax, and K* for mag-indo-1 dye were 0.19, 1.90, and 327 µM, respectively (n = 718 cells).
Data Analysis
Amperometric recordings were semiautomatically analyzed using software written in Igor (WaveMetrics). Some recordings with a small number of amperometric signals were plotted on a fast chart recorder and events were counted manually. The rate of exocytosis was defined as the number of amperometric spikes per 30-s time bin. To evaluate relative exocytosis, the rates of exocytosis in control and test conditions were averaged for 2 min. As the maximal exocytosis is reached at different times in different test solutions, the 2-min analysis period was taken after various time delays: 3 min for forskolin or PMA on 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)loaded cells (see Fig 4, and Fig 6 Fig 7 Fig 8), Rp-8-Br-cAMPS (see Fig 6) or BIS (see Fig 8) treatments, 2 min for forskolin, PMA or Sp-8-Br-cAMPS on cells not loaded with BAPTA (see Fig 5, Fig 6, and Fig 8), and 1 min for 2 mM Ca2+ on ionomycin- (see Fig 2) and hyperosmotic solution-treated (see Fig 10) cells. During treatments with hyperosmotic solution, only the last minute of the recordings was analyzed. Relative exocytosis was defined by the ratio of exocytosis rates after and before treatments.
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Kinetics of single quantal events were fitted with the sum of two exponential functions using events without a foot. Rise times (1090% of peak amplitude) and fall times (9010% of peak amplitude) were extracted from the fitted curves. Half-width was measured as time spent above 50% of peak amplitude.
All numerical values are given as mean ± SEM. The significance of differences between mean values of two groups was tested by Student's t test. The Kolmogorov-Smirnov test (see Fig 5) was performed using software written in Igor. A probability of P 0.05 was considered significant.
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RESULTS |
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Quantal Events Resemble Those of Excitable Cells
We begin by describing the amperometric signature of exocytosis in epithelial cells for comparison with better-characterized systems. Touching a polarized carbon-fiber electrode gently to a single cell previously loaded in 70 mM dopamine or serotonin revealed spontaneous amperometric current spikes (Fig 1A and Fig B). They reflect rapid oxidation of many thousands of oxidizable molecules released abruptly at the cell surface. In tests on five different batches of cells, signals were not observed without preloading the amine, indicating that the spikes represented the exogenous oxidizable amine. It was necessary to incubate epithelial cells in the 70-mM loading medium for longer than 30 min to detect the oxidation currents. Attempting to load with 1.5 mM of dopamine or serotonin for 4 h elicited much smaller peaks (not shown). Rates of spontaneous exocytosis ranged from 2 to 53 events per min (mean: 17.2 ± 4.9 events/min, n = 11 cells). The rate typically decreased over 45 h after loading was stopped, presumably due to a loss of vesicular amines through spontaneous exocytosis and by leak of cytoplasmic amines to the culture medium.
As shown in Fig 1 B, each spike had the rapid onset and decay that are characteristic of exocytotic release from single secretory vesicles in other systems (
Preceding large events with a good signal-to-noise ratio (Fig 1 B), one could occasionally see a small step of release preceding the main event, named the "foot" by 103,000 electronic charges per event. For a two-electron oxidation of dopamine, this would correspond to
51,500 molecules per spike. The similarities with amperometric studies in other cells argue that these amperometric spikes represent quantal exocytosis of loaded amines and that release begins with formation of a fusion pore (see DISCUSSION). However, one difference here is the extreme range of sizes of the amperometric events. Since our loading and recording methods did not isolate a specific class of secretory vesicles (
Ca2+ Stimulates Exocytosis
As for neurons and many endocrine cells, exocytosis in the pancreatic duct epithelial cells could be stimulated by elevating [Ca2+]i. We used the Ca2+ ionophore, ionomycin, to bring Ca2+ into the cytoplasm. Dopamine-loaded cells were incubated in Ca2+-free solution containing 5 µM ionomycin for 510 min. Subsequent application of a solution containing 2 mM Ca2+ induced vigorous exocytosis (Fig 2A and Fig B). The significant rise of the current baseline during the Ca2+ application very likely reflected spillover of released dopamine from regions not directly under the electrodeon the same cell and on neighboring cells. The mean rate of exocytosis increased to 9.6 ± 2.4 (n = 16) times the control rates. The barrage of exocytotic events seen at the end of the Ca2+ application in Fig 2 A was not seen in other similar experiments. It may reflect the sudden rise of [Ca2+]i late in the Ca2+ application (Fig 2 C).
We wanted to estimate the dependence of exocytosis on the mean [Ca2+]i achieved with ionomycin treatment. Initial experiments with indo-1 had shown that application of 2 mM Ca2+ in the presence of ionomycin raised [Ca2+]i into the range where the response of indo-1 saturates. Therefore, we switched to mag-indo-1-AM, a dye with much lower Ca2+ affinity. In various cells exposed to 2 mM Ca2+, [Ca2+]i increased monotonically, after a delay of 1 min (Fig 2C and Fig D). The average [Ca2+]i reported by this dye at the end of the 3-min Ca2+ stimulation was 313 ± 74 µM (n = 8). Although [Ca2+]i rose for several minutes, exocytosis developed early, before a large change in the mag-indo-1 signal was observed, indicating that exocytosis can be efficiently induced at low micromolar concentrations of Ca2+ (Fig 2 D). To improve our estimate, we examined exocytosis in ionomycin with several lower Ca2+ concentrations in the bathing solution (Fig 3A and Fig B). The 1 mM Ca2+ was about as effective as 2 mM, and 300 µM elicited a half-maximal rate of exocytosis. After these 3-min incubations in 5 µM ionomycin, the [Ca2+]i was definitely not proportional to extracellular [Ca2+]. With 1 mM Ca2+ outside, the [Ca2+]i rose to 107 ± 25 µM (n = 9), whereas with 300 and 100 µM outside, it reached only 419 ± 41 nM (n = 5) and 176 ± 13 nM (n = 6), respectively. (The first value was measured with mag-indo-1 and the latter two with indo-1.) Apparently, cellular Ca2+ clearance mechanisms remained relatively effective with the lower bath [Ca2+], and they seemed to become overwhelmed if [Ca2+]i rose above 5 µM for many seconds. This result led us to use higher concentrations of ionomycin, longer incubations, and Ca2+ clearance inhibitors when calibrating the dyes (see METHODS). In any case, exocytosis from the pancreatic epithelial cells is quite sensitive to Ca2+ since it reaches significant levels already with [Ca2+]i as low as 300400 nM (Fig 3 C).
Other Signaling Systems Elevate [Ca2+]i
Secretion from many gastrointestinal and airway epithelia can be stimulated by hormones and drugs that elevate intracellular levels of cAMP or stimulate phospholipase C. In cultured dog pancreatic epithelial cells, mucin secretion is induced by treatments that elevate cAMP (
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cAMP Induces PKA-dependent and [Ca2+]i -independent Exocytosis
Fig 4 A illustrates a typical long-lasting amperometric recording before and after activation of adenylyl cyclase by forskolin in a BAPTA-loaded cell. After 3 min of control recording, 20 µM forskolin was applied and the frequency of amperometric spikes gradually increased. The number of events in every 30-s interval is plotted in Fig 4 B. The frequency rose from near 6/min in control to 32/min after forskolin treatment, a fivefold increase. The activation was first apparent at 3060 s, and grew even after the forskolin was removed. The simultaneous [Ca2+]i measurement (Fig 4 C) confirmed that preloading with BAPTA had effectively eliminated any [Ca2+]i transient during the forskolin treatment.
The time course of individual quantal events was not changed by forskolin treatment. Thus, the average half-width of events was 4.9 ± 0.2 ms (204 events from 10 experiments, at a filter frequency of 500 Hz; P = 0.89 compared with the value of control events). The average 1090% rise- and fall-times were 1.13 ± 0.04 and 6.7 ± 0.3 ms, respectively. The mean current amplitude and charge of quantal events were slightly increased by forskolin treatment (1.11 ± 0.07 pA (P = 0.04) and 17.9 ± 2.1 fC (P = 0.39), respectively, n = 15 cells). Forskolin increased the rate of release of quanta of all sizes, but selectively enhanced the release of larger quanta, as can be seen in the histograms of Fig 5 that compare the fraction of events in each charge range. (A significance of P = 0.002 was calculated with the Kolmogorov-Smirnov test using a cumulative histogram of the same data.)
The effects of forskolin on cells with and without BAPTA loading are summarized in Fig 6 A. The amperometrically detected exocytosis rate in forskolin was 3.4 ± 1.2 (n = 8) relative to controls in BAPTA-loaded cells and 3.4 ± 0.6 (n = 23) in BAPTA-free cells. The basically identical effects of forskolin with or without buffering of the small transient rise of [Ca2+]i (Table 1) indicated that forskolin-stimulated exocytosis was mainly activated by cAMP. In parallel, we tested other agents known to increase intracellular cAMP levels and mucin secretion in these pancreatic epithelial cells (
Does the stimulation of exocytosis require activation of cAMP-dependent protein kinase (PKA)? As a test, we compared the actions of the Rp and Sp enantiomers of 8-Br-cAMPS (Fig 6 B). The Rp isomer is a specific inhibitor of PKA, and the Sp isomer is an activator. Application of Rp-8-Br-cAMPS did not change the rate of exocytosis in unstimulated cells (1.06 ± 0.15, n = 6), but it blocked the effect of forskolin applied 5 min later (0.80 ± 0.11, n = 6). In contrast, application of Sp-8-Br-cAMPS increased exocytosis by itself (2.7 ± 0.3, n = 9). These actions further support the hypothesis that stimulation of exocytosis is mediated through cAMP and PKA.
PKC also Stimulates [Ca2+]i -independent Exocytosis
We then tested the phorbol ester PMA to ask whether activating PKC also increased amperometrically detected exocytosis. The elevation of [Ca2+]i by PMA is minimal (Table 1). Nevertheless, we preloaded one batch of cells with BAPTA and tested the actions of 100 nM PMA. The phorbol ester increased exocytosis (Fig 7A and Fig B) without any accompanying change of [Ca2+]i (Fig 7 C; and Table 1), but the potentiation of exocytosis was smaller than that by cAMP. On average, this concentration of PMA elevated relative exocytosis to 2.7 ± 0.6 (n = 5) in cells preloaded with BAPTA and to 2.4 ± 0.4 (n = 13) without BAPTA (Fig 8 A). These values are not statistically different (P = 0.67). Next, we did controls to check whether PMA acted via PKC to increase exocytosis. A structurally related but inactive compound, 4--phorbol-12,13-didecanoate (4-
-phorbol) slightly increased exocytosis (1.37 ± 0.27, n = 5) at a concentration of 100 nM; this increase, however, was not statistically significant (P = 0.12). To reduce possible nonspecific effects, the concentration of the active and control phorbol esters was decreased to 10 nM (Fig 8 B). Exocytosis was still significantly evoked by PMA at 10 nM (2.2 ± 0.4, n = 4) but not by 4-
-phorbol (0.95 ± 0.21, n = 4). The PKC-selective inhibitor, BIS (500 nM), slightly reduced exocytosis by itself (0.68 ± 0.07, n = 10). After a 5-min incubation with BIS, PMA was not able to evoke exocytosis (0.44 ± 0.07, n = 9). The further decrease of exocytosis during the combined application of BIS and PMA may be due to a continued slow development of the effect of BIS.
With PKA and PKC Blocked, Exocytosis Is Induced Only by High [Ca2+]i
Using simultaneous measurements of [Ca2+]i and exocytosis, we have demonstrated that PKA and PKC can increase exocytosis when [Ca2+]i rises are blocked with BAPTA. Now we asked if the reverse is true. Can Ca2+ induce exocytosis when protein kinases are blocked? In these experiments, we use BIS to block PKC as before, and H-89 to block PKA. (The expense of using Rp-8-Br-cAMPS would have been too great.) To check the efficacy of H-89 in blocking PKA, we asked whether pretreatment with 10 µM H-89 for 5 min reduced relative exocytosis induced by 10 µM forskolin. In untreated cells, forskolin raised relative exocytosis from 3.31 ± 0.85 (n = 3), and in H-89treated cells, forskolin was ineffectual (1.15 ± 0.22, n = 7, relative to H-89 alone).
Both kinase inhibitors depressed relative exocytosis in control experiments. It was reduced to 0.64 ± 0.05 by H-89 (n = 27) and to 0.76 ± 0.06 (n = 24) by BIS (Fig 9 A). The depression implies either that there is some background activation of the kinases in unstimulated cells or that the inhibitors have nonspecific actions at other steps in exocytosis. Therefore, in the statistical tests below, we use the H-89- or BIS-inhibited measurement as the control value for calculating the effect of subsequently added Ca2+.
Next, tests were done on the effects of [Ca2+]i elevation while kinases were inhibited. The first tests used 300 µM extracellular Ca2+, which raises [Ca2+]i only to submicromolar concentrations (400 nM). The low Ca2+ stimulus applied to uninhibited, ionomycin-treated cells increased relative exocytosis to 3.01 ± 0.37 (Fig 9 A). The same stimulus was not effectual on cells treated with 10 µM H-89 (0.71 ± 0.21, n = 8, relative exocytosis compared with H-89 alone) or with 500 nM BIS (1.08 ± 0.26, n = 16, relative exocytosis compared with BIS alone). Thus, protein kinase inhibitors block the effects of small [Ca2+]i elevations. Then tests were repeated with 2 mM extracellular Ca2+, which raises [Ca2+]i to 300 µM. The exocytosis induced by this stimulus was not significantly reduced by inhibition of PKA or PKC or by simultaneous inhibition of both kinases (Fig 9 B).
Some Properties of Exocytosis Differ Between Epithelial and Excitable Cells
In the beginning of the RESULTS, we emphasized similarities between the secretion in epithelial cells and that of excitable cells. There are some clear differences, however: insensitivity to hypertonic shocks, insensitivity to KCl depolarization, and occasional giant multi-vesicular secretory events.
Synapses release a small number of neurotransmitter quanta upon sudden stimulation with hypertonic solutions (
In excitable cells, exocytosis is normally stimulated by membrane depolarization, which allows Ca2+ to enter cells via voltage-gated Ca2+ channels. As exocytosis from epithelial cells is also Ca2+ sensitive, it was interesting to test whether they can use a voltage-gated Ca2+ influx mechanism. The resting potential of the cultured epithelial cells was relatively low, -39 ± 2.1 mV (measured under perforated-patch conditions). It depolarized to -21 ± 3.1 mV (n = 11) when all the extracellular NaCl was replaced by KCl. However, the KCl depolarization did not change the rate of exocytosis (exocytosis relative to control = 0.97 ± 0.07, n = 9), indicating that, at least in this potential range, these cells lack voltage-gated Ca2+ entry mechanisms.
We occasionally observed large, long-lasting amperometric events. These complex events were characterized by multiple overlapping peaks, suggesting compound exocytosis with overlapping fusion of a large number of vesicles (Fig 11). As an empirical criterion to detect such events, we used a sliding window of 50 adjacent sample points. A large event was scored if the total charge in this interval exceeded 20x the average charge of a single quantal event. The window was moved by 25 points each time to ensure the detection of the complex events. By this criterion, there were 12 giant events out of 2,714 amperometric events (one per 226 events) in seven experiments. The occurrence may be overestimated, as only recordings containing at least one obvious complex event were included in this analysis. The average charge of such complex amperometric events was 99 ± 25 (n = 7) times that of the quantal events. Such events were seen both during stimulated release and during spontaneous release in unstimulated cells.
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DISCUSSION |
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Secretion in Epithelia
The physiological ion transport and exocytosis in epithelia are regulated by hormones and neurotransmitters through stimulation of either adenylyl cyclase or phospholipase C. In most studies, it has not been possible to measure or regulate possible intracellular Ca2+ transients in response to the hormones. For hormones acting via phospholipase C, a Ca2+ transient as well as activation of PKC would be expected; all these pathways stimulate mucin secretion (
Secretion of mucin has previously been demonstrated in confluent monolayers of the cultured dog pancreatic epithelial cells used here. VIP and epinephrine raised the cAMP content of these differentiated cells approximately sixfold, and 4-h incubations with VIP, epinephrine, or dibutyryl cAMP increased mucin secretion to 1.53x control amounts (
We provide further evidence that stimulation by cAMP requires activation of PKA and does not require a [Ca2+]i elevation, and that PKC also will stimulate exocytosis without a [Ca2+]i elevation. Finally, high levels of [Ca2+]i alone can evoke exocytosis without activation of PKA or PKC. However, exocytosis evoked by submicromolar Ca2+ was significantly reduced when the protein kinases are pharmacologically inhibited. Such results suggest that Ca2+ induces exocytosis by different pathways depending on its concentration, but a definitive conclusion awaits further study with other types of protein kinase inhibitors or cells lacking PKA or PKC expression.
Comparison of Epithelia and Excitable Cells
The secretory mechanisms of excitable cells and epithelia have similarities and differences. Our study may be the first to apply amperometry to epithelial secretion. This allows an improvement of the time resolution of several orders of magnitude and resolution of single-vesicle release events. As in excitable cells, we find that the basic release event has a rise time shorter than 1 ms and is preceded, for some events, by a foot lasting several milliseconds. It appears, therefore, that, as in mast cells or chromaffin cells (7 ms and half widths of 5 ms. This lies between the observed 515-ms half widths for catecholamine release from mammalian chromaffin cells (
What are the differences from excitable cells? As they apparently lack voltage-gated calcium channels, depolarization does not stimulate exocytosis in epithelial cells as it does in excitable cells. Additionally, hypertonic solutions depress exocytosis in our cells and in chromaffin cells (
All of these functional differences may point to well-known structural factors. In epithelia, although many secretory granules may be near the plasma membrane (
Protein-Kinase Sensitivity of Exocytosis in Neurons and Endocrine Cells
Ca2+ is very clearly the immediate trigger of evoked transmitter release at synapses. Nevertheless, pharmacological activation of PKA or PKC increases evoked and spontaneous excitatory and inhibitory postsynaptic currents two- to fourfold, much as in epithelial cells. For example, phorbol esters acting via PKC increase the frequency but not the amplitude of spontaneous GABA release from interneurons and glutamate release from hippocampal neurons (
Which Vesicles Are Being Reported?
Our loading method does not allow selective study of a specific class of secretory vesicle in the way that a biochemical or postsynaptic current assay would. With the high serotonin and dopamine concentrations used here, loading does not depend on monoamine transporters (
We can compare the number of oxidizable molecules released per quantum in different cells. In forskolin-stimulated pancreatic duct cells, we see a broad distribution of sizes and a mean of 100,000 elementary charges, corresponding to
50,000 dopamine molecules per vesicle, but with 20% of the events < 7,000 molecules and 7% > 100,000 molecules (Fig 5). In the same kind of assay with cells containing endogenous catecholamine or serotonin, the approximate number of molecules released per quantum is 7 x 106 from chromaffin cells (
What are the giant complex events (Fig 11)? The hypothesis that they represent the death of a cell, with release of all oxidizable contents, can be excluded by two observations. First, if the cell membrane is deliberately ruptured by advancing the amperometric probe by several micrometers, a much larger oxidation current (peak amplitude >50 pA) of longer duration (>10 s) was observed. This reflects a considerable quantity of cytoplasmic monoamine. Second, the more typical quantal events continue unabated with fast rise and fall times immediately after a complex event, suggesting that the cell is still functional and that its membrane remains within 1 µm of the probe.
A more likely explanation for these giant complex events is that a large number of quantal events are superimposed. This would account for the clear spike-like substructure of the complex event. However, it is unlikely that complex events are a random superposition of independent quantal events. We can estimate the chance of that occurring. Since the average frequency of spontaneous quantal events is one per 3 s, the probability of observing a quantum within a 500 ms time window is 1/6. Provided that amperometric events are randomly distributed, the probability of having 100 or more independent events occurring in a 500-ms time window is (1/6)100. The probability of seeing a complex event within 500 ms window was actually 1/1,360, calculated as the probability of observing any event (1/6) multiplied by fraction of complex events (1/226). Therefore, some concerted process must coordinate multivesicular events. It might be a form of compound exocytosis in which some vesicles are fusing to each other before the final fusion to the plasma membrane or it may represent a period in which the probability of fusion has been raised by several orders of magnitude by some intracellular signal. The number of molecules released in one giant event exceeds several million, which is comparable with the release from a typical chromaffin granuleexcept that it is spread out in time so that the amplitude is not 100x the mean quantal amplitude. Compound exocytosis and also apocrine-like secretion are commonly invoked in morphological studies of stimulated, mucin secreting epithelia (
In conclusion, exocytosis in pancreatic duct epithelial cells shares several features with exocytosis in excitable cells. However, overall, the range of regulation, the speed of regulation, and the maximum rate of exocytosis are smaller. In the epithelium, the effects of kinases and Ca2+ are similar in magnitude, whereas in neurons the effect of Ca2+ is much stronger than that of kinases. Therefore, hormonal regulation via kinases becomes a viable way to regulate exocytosis from epithelia in the absence of any Ca2+ signal. Its molecular basis needs clarification.
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Footnotes |
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Portions of this work were previously published in abstract form (Koh, D.-S., M.W. Moody, T.D. Nguyen, B.L.Tempel, and B. Hille. 1997. Soc. Neurosci. Abstr. 22:467).
1 Abbreviations used in this paper: BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BIS, bisindolylmaleimide I; VIP, vasoactive intestinal peptide.
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Acknowledgements |
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We thank Drs. Ed Kaftan and Tao Xu for reading the manuscript and Drs. S.P. Lee and P. Verdugo for helpful discussions. We thank L. Miller and D. Anderson for technical assistance.
This work was supported by grants from the National Institutes of Health (AR17803), the Cystic Fibrosis Foundation (R565), and the Department of Veterans Affairs (Merit Review).
Submitted: 30 March 2000
Revised: 8 August 2000
Accepted: 9 August 2000
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