INTRODUCTION

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It is generally accepted that intracellular Ca²⁺ plays a crucial role in controlling exocytosis (1-5). In exocrine gland cells, an agonist-induced rise in the free cytosolic Ca²⁺ concentration ([Ca²⁺]_i)¹ (1) evokes exocytosis (6). Calcium pumps have been found in different regions of the plasma membrane of exocrine acinar cells (7), and the rise in [Ca²⁺]_i also activates Ca²⁺ ATPase-mediated extrusion across the plasma membrane (8-10). The secretory granules have a very high Ca²⁺ concentration (11, 12), and the exocytotic event itself must therefore also result in Ca²⁺ release. However, in previous studies on pancreatic acinar cells (10), where agonist-evoked secretion is severely reduced at a low extracellular Ca²⁺ concentration, we estimated that the major part of the Ca²⁺ extrusion occurring in response to supramaximal agonist stimulation was due to Ca²⁺ pump-mediated outflux across the plasma membrane rather than exocytosis (8-10).

Despite the generally accepted role for Ca²⁺ in regulating exocytosis, there are gland cells, for example the salivary glands, in which the major intracellular signal evoking exocytosis is not a rise in [Ca²⁺]_i but an increase in the cyclic AMP concentration (13, 14). Excitation of -adrenergic receptors stimulates protein secretion from salivary glands via cyclic AMP formation without any rise in [Ca²⁺]_i (13, 14). In these cells, the secretory response is hardly affected by the presence or absence of extracellular Ca²⁺ (15). Since salivary gland cells possess cholinergic muscarinic and -adrenergic receptors, which control [Ca²⁺]_i as well as -adrenergic receptors that regulate cyclic AMP formation (13, 14), we have compared Ca²⁺ extrusion responses to the muscarinic agonist acetylcholine and the -adrenergic agonist isoproterenol (ISP) from single cells or small salivary gland cell clusters.

With the help of the droplet technique (8, 9, 16) as well as a recently developed method for localizing Ca²⁺ extrusion (10, 17), we have obtained evidence indicating that ISP evokes calcium extrusion by exocytosis. In contrast, ACh evokes calcium efflux from the salivary glands, which is most likely mediated by both Ca²⁺ pumps and exocytosis. We have been able to detect Ca²⁺ extrusion apparently resulting from a single secretory event and have estimated the amount of Ca²⁺ secreted as a result of this process. Our results indicate that in the salivary glands exocytosis can be the main route for calcium extrusion from cells.

	MATERIALS AND METHODS

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Fragments of mouse or rat submandibular gland were digested by pure collagenase to obtain small cell clusters as described previously (18). In some experiments, where it is directly indicated under "Results," cells were loaded with the fluorescent Ca²⁺-sensitive indicator fura-red/AM or fura-2/AM (Molecular Probes) for 30 min at room temperature as described previously for fura-2 loading (19). In the confocal microscopy experiments, a group of either dye-loaded or unloaded cell clusters were placed in a small experimental chamber (approximately 200 µl) containing nominally Ca²⁺-free solution with the 30-70 µM Ca²⁺-sensitive dye, calcium green-1, bound to dextran (M_r 500,000, Molecular Probes). The free Ca²⁺ concentration in the extracellular solution under these conditions was around 0.2-0.4 µM. As there is a substantial difference between the emission spectra of calcium green-1 dextran and fura-red, it is possible to monitor simultaneously the intracellular and extracellular Ca²⁺ concentrations. The fluorescent signals from these intracellular and extracellular dyes were recorded using a Noran Odyssey confocal microscope (Noran Instruments) with an excitation wavelength of 488 nm and emission wavelengths of 530 and 650 nm for calcium green-1 dextran and fura-red, respectively. These fluorescence signals were acquired in several regions of interest. Boxes were placed in different areas within the confocal section, and averaged fluorescence signals were recorded from these boxes. In a few experiments, images of the clusters were taken (2 images/s) simultaneously with these recordings. All images were analyzed and processed by Two D Intervision analysis software (Noran Instruments). The detailed description of the technique of calcium efflux measurements using dextran-bound calcium indicators have recently been published (10, 17). In the experiments with the droplet technique (16), a single fura-2 loaded cluster was maintained in a small (20-50 cluster volumes) droplet containing nominally calcium-free solution and 100 or 150 µM of the calcium indicator fluo-3. The fluorescence of the cluster-droplet system was recorded with a SPEX (Glen Spectra) DM 3000 CM system using the excitation wavelengths 380 and 490 nm and the emission wavelength 530 nm. The amount of calcium extruded was calculated as the product of the increase in extracellular calcium concentration and the calculated volume of the microdroplet (16). To relate the amount extruded to the decrease in the total cellular calcium concentration, we divided the amount of calcium extruded by the calculated cell volume. The free intracellular calcium concentration was assessed from the fura-2 fluorescence as described previously (9, 16).

Similar results were obtained in droplet technique experiments with both rat (n = 10) and mouse (n = 4) submandibular cell clusters. The quantification of calcium fluxes in the section about droplet technique experiments is given for experiments on rat submandibular cells.

Agonist applications were made by either the direct addition of a small droplet of highly concentrated agonist stock solution or by iontophoresis from a microelectrode. Iontophoretic injection currents were from 10 to 100 nA.

The extracellular solution contained (in mM) NaCl (140), MgCl₂ (0.66-1.13), KCl (4.7), Hepes (10), glucose (10), pH 7.2, with NaOH. Ca²⁺ indicators were purchased from Molecular Probes (Eugene). All other chemicals were purchased from Sigma (Dorset, UK). Experiments were carried out at room temperature.

	RESULTS

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Measurements of ISP- and ACh-induced Ca Efflux with the Droplet Technique-- To quantitatively characterize the efflux of calcium from submandibular cells, we have used the droplet technique (16). Submandibular acinar cell clusters were placed in small droplets of extracellular solution supplemented with the calcium-sensitive dye fluo-3. In this series of experiments, cells were loaded with fura-2. The total amount of calcium extruded from the clusters was calculated on the basis of fluo-3 measurements (16). Clusters placed in the droplets revealed a substantial basal calcium efflux corresponding to a reduction of the total cellular calcium concentration of 81 ± 15 (S.E.) µM/min (n = 7) (rat submandibular cells).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Droplet technique measurements of calcium efflux from submandibular cells. Cells were loaded by fura-2. Extracellular solution contained fluo-3. Changes of [Ca]o and [Ca2+]i as a result of ISP stimulation. Panel a shows elevation of [Ca]o in droplet solution measured using fluo-3 (top trace) and measurement of [Ca2+]i (bottom trace) using fura-2. Panel b shows the rate of calcium extrusion calculated using the derivative of [Ca]o curve (16). Bars represent the time when ISP was present in droplet solution.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Droplet technique measurements of calcium efflux from submandibular cells. Cells loaded by fura-2. Extracellular solution contained fluo-3. Changes of [Ca]o and [Ca2+]i as a result of ACh stimulation. Panel a shows elevation of [Ca]o in droplet solution measured using fluo-3 (top trace) and [Ca2+]i (bottom trace) using fura-2. Panel b shows the rate of calcium extrusion. Bars represent the time when ACh was present in droplet solution.

Ca²⁺ Extrusion Spikes-- Ca²⁺ extrusion by Ca²⁺ pumps and Ca²⁺ efflux due to exocytosis should result in different types of [Ca²⁺]_o changes. Exocytosis might be expected to induce short Ca²⁺ spikes in the extracellular solution, when individual granules deliver their Ca²⁺ load, whereas pumps and exchangers should produce a "smooth" Ca²⁺ efflux. On the basis of these considerations, we made an attempt to distinguish the mechanisms that are involved in ISP- and ACh-induced calcium efflux using confocal microscopy.

View larger version (76K): [in this window] [in a new window]   Fig. 3.   Spatio-temporal pattern of exocytotic calcium extrusion from a small cluster of submandibular cells. Panel a. presents a transmitted light picture of a small cluster (five cells). Preliminary observation of the cluster revealed that there was just one place on its boundary (probably the place where the acinar lumen is in contact with the extracellular solution) where massive secretion under ISP stimulation took place (on this confocal section). This place is indicated by the box in the upper left part of the figure; bar corresponds to 10 µm. The temporal pattern of the fluorescence changes in this box is shown in panel b. The movie (c) was captured during the period indicated by the short bar in panel b and shows the spatio-temporal pattern of Ca2+-sensitive fluorescence changes in the box during the time indicated.

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Different Ca²⁺ Extrusion Responses to Isoproterenol and Acetylcholine-- In the next set of experiments, we mainly recorded the averaged [Ca²⁺]_o signals in the regions of interest. Signals recorded in such experiments depend on the size of the boxes, the position of the boxes with respect to the site of calcium efflux, as well as on the amount of calcium extruded.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Calcium extrusion responses induced by ISP application. [Ca2+]o was measured using calcium green-1 dextran. Application of ISP is shown by the bar.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   ACh-induced calcium signals. ACh was applied by iontophoretic injection. Bar represents the duration of ACh application. Two types of ACh-induced responses in the extracellular solution are shown: combination of smooth components and small spikes (top curve) and smooth response (bottom curve).

	DISCUSSION

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In this paper we have demonstrated a substantial release of Ca²⁺ from submandibular salivary gland cells evoked by ISP stimulation of -adrenergic receptors, without any apparent rise in [Ca²⁺]_i. We are proposing that the ISP-evoked Ca²⁺ release occurs as a consequence of exocytosis. The main points arguing in favor of our conclusion that ISP evokes Ca²⁺ release due to exocytosis rather than Ca²⁺ pump activation are as follows. 1) The calcium release induced by ISP is spiking. 2) ISP does not evoke any measurable rise in [Ca²⁺]_i, and there is therefore no reason to expect Ca²⁺ extrusion by Ca²⁺ ATPase activation. 3) Although ISP does not increase [Ca²⁺]_i, the Ca²⁺ extrusion induced is larger than that seen in response to ACh stimulation that does substantially elevate [Ca²⁺]_i. 4) ISP is a more potent secretagogue in the salivary glands than ACh since it induces a more substantial and sustained secretion of protein than can be achieved by ACh (13, 14, 15, 26, 27). 5) Secretory granules from many different types of gland cells, including submandibular salivary gland cells, have a high concentration of calcium (about 10-100 mM) (11, 12). Exocytosis must therefore result in an elevation of [Ca²⁺]_o. 6) The Ca²⁺ efflux responses induced by ISP and ACh seem to occur from different sources, since ISP could still evoke a substantial Ca²⁺ extrusion following an ACh stimulation sufficiently long to discharge the internal Ca²⁺ store. In complementary experiments, a long period of ISP incubation did not prevent ACh induced efflux.

By using a high resolution imaging technique, we have been able to obtain shortlasting Ca²⁺ extrusion spikes particularly during ISP, but also with ACh stimulation. Although ISP is known to be the most potent secretagogue, ACh can also evoke protein secretion, and it is therefore simplest to explain these extracellular Ca²⁺ spikes as consequences of single exocytotic events that may, however, involve several granules (compound exocytosis). The total calcium efflux response induced by ACh is, therefore, a combination of Ca²⁺ extrusion by Ca²⁺ pumps of plasma membrane and calcium efflux mediated by exocytosis. It is difficult to evaluate the contribution of these two components in ACh-induced efflux, but the relatively few events that overlay large calcium transients in experiments with calcium green-1 dextran (Fig. 5), the very good correlation between the rate of calcium extrusion and intracellular Ca²⁺ concentration recorded in the droplet experiments (Fig. 2), and the fact that calcium efflux can be evoked by ACh after extended incubation with ISP suggest that Ca²⁺ pumps make the major contribution to ACh-induced calcium efflux. On the contrary, exocytosis is most likely the major mechanism responsible for the calcium efflux induced by ISP in the salivary gland acinar cells.

The basal spiking activity can constantly extrude a considerable amount of calcium. Our previous results have shown that under analogous conditions (room temperature, low extracellular calcium concentration) pancreatic acinar cells revealed practically no secretion. In experiments with pancreatic acinar cells placed in a solution containing a dextran-bound calcium indicator, we have not seen fast calcium transients in external near-membrane regions. In pancreatic acinar cells, the calcium efflux measured in droplet experiments using dextran-bound calcium indicators was due to activity of the calcium pumps of the plasma membrane (8, 10, 16). In these cells, the rate of basal calcium extrusion measured by the droplet technique was approximately 8 µM/min, whereas in the submandibular cells, it is about 10-fold higher (81 ± 15 µM/min). We mainly attribute this difference to the higher rate of basal exocytosis in submandibular cells compared with pancreatic acinar cells. It once again means that exocytosis could be one of the main mechanisms balancing intracellular calcium content.

In many cell types, the exocytosis is a virtually permanent process during the whole life of a cell. It seems reasonable to suggest that cells can sequester some substances (like Ca²⁺), which are planned to be expelled from the cells, inside the secretory granules and secrete them together with the main secretory products like proteolytic enzymes or neurotransmitters. This would have the following advantages: (i) energy spent to fuse secretory vesicles (that in any case has to be spent to secrete the main secretory products) will also be used to extrude other substances; (ii) most of the calcium ions that enter the cytosol are sequestered into intracellular stores at the first step (28, 29). To extrude Ca²⁺ by plasma membrane systems would necessitate release to the cytosol first, and only then could Ca²⁺ be extruded to the extracellular medium. This would mean double pumping of each Ca²⁺ against high gradients, resulting in energy wastage; and (iii) the whole surface area of intracellular compartments is substantially larger than the area of the plasma membrane. This gives room for accommodating more Ca²⁺-regulating systems (like Ca²⁺ pumps) that will take up Ca²⁺ inside the intracellular compartment. Including Ca²⁺ into secretory vesicles will eventually lead to extrusion into the extracellular environment.

Ca²⁺ can be sequestered in secretory granules both directly via Ca-ATPases and/or the Ca/H exchanger (11, 30, 31) and indirectly via uptake of calcium accumulated into internal calcium stores (28, 32, 33). Regardless of the mechanism by which Ca²⁺ is sequestered in the secretory granules, their calcium content is very high, approaching 100 mM in some cell types (11). Such a high total calcium concentration results in a massive calcium extrusion during even a single secretory event. For example, exocytosis of one secretory granule (assume that a granule equals 10⁴ of the cell volume) will decrease the total intracellular calcium concentration by 1-10 µM, which is a substantial change in comparison to the free intracellular calcium concentration (0.1 µM). Besides, it has been shown or suggested that a high intragranular calcium concentration is important for processing secretory products, protein packaging, and granular fusion with the plasma membrane (34, 35).

Our results strongly suggest that the scheme of calcium extrusion described above could take place in many types of cells that undergo exocytosis. It has been proposed that exocytosis could be a possible export route for calcium from bovine adrenal medullary cells (36), neurohypophyseal nerve endings (37), and sea urchin eggs (38, 39). The present work characterizes for the first time both the amount and the kinetics of exocytotic calcium efflux measured at the single cell level and compared with calcium efflux due to plasma membrane calcium pumps. We have found that calcium release via exocytosis may be a route for calcium extrusion, and in some cases, the main part of the calcium efflux occurs by means of this mechanism.