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. 2000 Feb 1;522(Pt 3):403–416. doi: 10.1111/j.1469-7793.2000.t01-1-00403.x

Carbachol-induced [Ca2+]i increase, but not activation of protein kinase C, stimulates exocytosis in rat parotid acini

Keiichi Yoshimura *, Masataka Murakami *, Akihisa Segawa †
PMCID: PMC2269767  PMID: 10713965

Abstract

  1. A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)-induced amylase secretion.

  2. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose-response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i).

  3. The CCh-induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3-sensitive stores was relatively ATP independent.

  4. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca2+-free medium evoked a second rapid and large peak of amylase secretion.

  5. Phorbol 12,13-dibutyrate (PDBu) potentiated the CCh-induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh-induced [Ca2+]i changes.

  6. PKC inhibitors such as Ro 31–8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone.

  7. These results suggest that a CCh-induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP-dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh-induced sustained plateau of amylase secretion. PDBu-induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

Amylase secretion from rat parotid acinar cells is regulated by at least two distinct mechanisms (Butcher & Putney, 1980; Putney, 1986). Activation of the β-adrenergic receptor by isoproterenol (isoprenaline), the effect of which is mediated by cAMP, evokes a large increase in the rate of amylase secretion. This pathway of secretion is little decreased by the removal of extracellular Ca2+ and appears to be independent of the cytosolic free Ca2+concentration ([Ca2+]i). Another mechanism to stimulate amylase secretion is activated by Ca2+-mobilizing agonists such as muscarinic cholinergic, α-adrenergic and substance P receptor stimulants. Since the removal of extracellular Ca2+ causes a large reduction in amylase secretion induced by these agonists (Putney et al. 1977; Watson et al. 1979; Yoshimura et al. 1984), changes in [Ca2+]i have been assumed to be crucial to the effects of these agonists. However, the precise role of [Ca2+]i in the regulation of amylase secretion has still not been fully elucidated.

Stimulation with Ca2+-mobilizing agonists activates phospholipase C, resulting in the production of InsP3 and 1,2-diacylglycerol (DAG) (Berridge & Irvine, 1984). InsP3 stimulates mobilization of Ca2+ from InsP3-sensitive stores and the entry of Ca2+ across the plasma membrane, while DAG activates protein kinase C (PKC). Previous studies have shown that carbachol (CCh) produces biphasic changes in the cytosolic free Ca2+ concentration ([Ca2+]i), with an initial rapid and large peak followed by a sustained plateau, in rat parotid acinar cells (Merritt & Rink, 1987). However, it is not known how the biphasic changes in [Ca2+]i and the activation of PKC induced by CCh are related to the regulation of amylase secretion. Tojyo et al. (1992) suggested that the activation of PKC, but not the rise in [Ca2+]i, is mainly responsible for the regulation of CCh-induced amylase secretion.

We have developed a method for the perfusion of isolated rat parotid acinar cells by embedding them in Bio-Gel P-2 resin. Compared with the conventional batch measurement technique, our perfusion system provided much better time resolutions of amylase secretion. Using this system, we found that CCh evokes biphasic changes in amylase secretion with an initial large but transient peak and a following sustained plateau (Yoshimura & Nezu, 1991). The time profiles of amylase secretion induced by CCh are very similar to those of [Ca2+]i response, suggesting changes in [Ca2+]i are crucial to the regulation of amylase secretion. In addition, the magnitude of the CCh-induced maximum response of the initial peak was comparable with that of isoproterenol, suggesting CCh has a very large intrinsic activity to stimulate amylase secretion. Thus, our results obtained by using perfused parotid acinar cells were quite different from the general perspective that the Ca2+-mobilizing agonists have a very limited effect in stimulating amylase secretion.

In the present experiments, we used our perfusion system to ascertain which of the two, [Ca2+]i and PKC, is the more dominant regulator of CCh-induced amylase secretion. One major conclusion from our studies is that the CCh-induced rapid rise in [Ca2+]i that is released from InsP3-sensitive stores, but not the activation of PKC, is mainly responsible for the CCh-induced initial peak response; the major effect of Ca2+ is to trigger the final fusion and/or exocytosis of primed secretory granules. CCh also has some intrinsic, but unknown, mechanisms for priming the secretory granules, which in collaboration with Ca2+ influxed into cells, produces the CCh-induced sustained plateau response. Activation of PKC by PDBu potentiates CCh-induced amylase secretion by promoting the priming of the secretory granules, thereby enhancing the efficacy for Ca2+ to evoke final fusion/exocytosis.

METHODS

Preparation of parotid acinar cells

All experiments were performed in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (1998). Animals were killed immediately by cervical dislocation. Isolated parotid acinar cells were prepared from non-fasted male Wistar rats (weighing 250–380 g) by collagenase and hyaluronidase digestion procedures described previously (Yoshimura & Nezu, 1991, 1992). The isolated acini were finally suspended in 5 ml of medium containing 1 % bovine serum albumin and 0.5 mg ml−1 trypsin inhibitor and kept at room temperature with gentle shaking until the start of each perfusion. The incubation medium contained (mM): NaCl, 127; KCl, 4.0; MgCl2, 1.2; CaCl2, 1.0; potassium phosphate, 1.0; Hepes, 20; glucose, 10; glutamate, 5; and 0.1 % bovine serum albumin at pH 7.4.

Perfusion of parotid acinar cells

Column perfusion of parotid acinar cells was performed as described in our previous papers (Yoshimura & Nezu, 1991; Yoshimura et al. 1998). In brief, acini (about 1 mg of cell protein) mixed with 200 μl preswollen Bio-Gel P-2 resin (fine, 45–90 mm), were layered onto 80 μl of the gel packed in 1 ml micropipette tips. The cells were perfused at 37°C with the medium bubbled with 100 % O2 at a flow rate of 1 ml min−1. Every 30 s a fraction (unless indicated otherwise) was collected for measurement of amylase activities. After each perfusion, cells in the column were lysed by homogenization with a Polytron (Kinematica; at a speed of 6 for 20 s) in 20 mM Mops buffer, pH 6.9, containing 0.1 % Triton X-100. The homogenates were centrifuged for 5 min at 900 g and the supernatant was saved. Amylase activities in each fraction and in the supernatant were assayed by the method of Bernfeld (1955) with a slight modification in that Mops buffer was used instead of potassium phosphate buffer. When amylase activity in the elutes from a Ca2+-free medium containing 0.2 mM EGTA was assayed, 0.5 mM Ca2+ was added to the buffer. The total amylase activity remaining in the cells at the beginning of each time point was calculated. The rate of amylase secretion at each time was expressed as a percentage of the total amylase activity (fractional amylase release, % min−1). While the time profiles of the rate of amylase secretion with each agonist were consistent, the magnitude of the response to the same agonists varied greatly depending on the preparation. The effects of various treatments on the response to each agonist, therefore, were compared with cell columns prepared from the same cell suspensions; the results obtained in these conditions were analysed by Student's paired sample t test. All results are quoted as means ±s.e.m. In some experiments, when several cell columns were prepared from each batch of the cell suspensions, the order of experiments was randomly changed. The interval between the onset of the first cell column to the last was usually less than 60 min. The magnitude of the response to agonists at 60 min after preparation were not different from those immediately after preparation. However, the basal (non-stimulated) rate of amylase secretion sometimes increased gradually with time and became, at most, about 50 % higher than the first.

Stock solutions of phorbol esters and ionomycin were prepared at a concentration of 5 mM in DMSO and 10 mM in ethanol, respectively, and stored at −20°C. The final concentrations of DMSO and ethanol in diluted solutions were 0.02 %. Ro 31–8220 and staurosporine were prepared as 10 mM and 500 μM stock solutions, respectively, in DMSO.

Measurement of [Ca2+]i in cell suspensions

Isolated rat parotid acinar cells were incubated with 2 μM fura-2 acetoxymethyl ester (fura-2 AM) for 45 min at 37°C, washed twice, and suspended in 10 ml of the buffer containing 1 % bovine serum albumin and 0.5 mg ml−1 trypsin inhibitor. The cells were kept at room temperature in the dark with gentle shaking. Just before use, 1 ml aliquots of the cell suspensions were centrifuged and resuspended in fresh media (2 ml) at 37°C in plastic cuvettes. The fura-2 fluorescence of the cells was recorded by a Hitachi 4010 spectrofluorometer as described previously (Yoshimura & Nezu, 1991).

Materials

Collagenase, type II, was purchased from Sigma Chemical Co. or Worthington Biochemical Co.; carbachol (carbamylcholine chloride, CCh), phorbol 12,13-dibutyrate (PDBu), hyaluronidase, thapsigargin and trypsin inhibitor were purchased from Sigma Chemical Co.; staurosporine was from Wako Chemical Co.; 4α-phorbol 12,13-dibutyrate (4α-PDBu) was from Funakoshi Co.; ionomycin was from Calbiochem; fura-2 AM and BAPTA AM were from Dojindo Lab. and Lucifer Yellow (LY) was from Molecular Probes. Bio-Gel P-2 gel was from Bio-Rad Laboratories. Ro 31–8220 was kindly supplied as a gift from Dr D. Bradshaw of Roche Products Limited.

RESULTS

Characteristics of CCh-induced amylase secretion in perfused parotid acinar cells

The basal (non-stimulated) rate of amylase secretion, as determined by our perfusion system, was 0.16 ± 0.04 % min−1 (n = 7; mean ±s.e.m.). CCh (1 μM to 1 mM) evoked biphasic stimulation of amylase secretion with an initial rapid and large but transient peak. This was followed by a sustained plateau that persisted until CCh was removed. The initial peak response induced by 100 μM CCh was 1.46 ± 0.20 % min−1 (n = 7), about ten times the resting level. The sustained plateau response, which was calculated as a mean between 4 and 5 min, was 0.26 ± 0.04 % min−1, about 15 % of the initial peak (Fig. 1Aa). Concentration-response analysis of the initial peak induced by CCh shows that 0.1 and 100 μM CCh evoked the minimum and the maximum responses, respectively. On the other hand, it required about 10 μM CCh to evoke the maximum response of the sustained plateau. The EC50 for CCh-induced initial peak was about 5 μM (Fig. 1B).

Figure 1. Time course and concentration-response of amylase secretion induced by CCh.

Figure 1

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Parotid acinar cells embedded in Bio-Gel P-2 were packed in small columns and stimulated with various concentrations (0.1 μM to 1 mM) of CCh by using the perfusion system. Aa, time profiles of amylase secretion induced by various concentrations of CCh. The order of the start of perfusion was changed as follows. Four cell preparations were first perfused from higher concentrations of CCh, and three cell preparations were perfused from lower concentrations of CCh. Ab, time profiles of CCh (100 μM)-induced initial changes in amylase secretion were examined in more precise time resolutions. Lucifer Yellow (0.2 mg (10 ml)−1) was added simultaneously with CCh to monitor the concentration of CCh in the column. Fractions were collected every 6 s. Amylase activity and fluorescence of LY (excitation, 462 nm; emission, 526 nm) in each fraction were determined. A typical trace of three other independent cell preparations is shown. B, summary of concentration-responses of the CCh-induced initial peak and sustained plateau of amylase secretion. Means of the results obtained from seven independent cell preparations are shown with their standard errors. Where error bars are invisible, they are masked by the data points.

To obtain more detailed information on CCh-induced initial changes of amylase secretion, Lucifer Yellow (LY) was added to the perfusates simultaneously with CCh (100 μM). Fluorescence of LY in each fraction was used as an index of the concentration of CCh. The concentration of CCh in the column increased linearly, and it required about 1 min to reach the concentration of applied CCh. The stimulative effect of CCh on amylase secretion was very rapid, and hence we were not able to detect any observable time lag for CCh to stimulate amylase secretion (Fig. 1Ab). Furthermore, the initial peak response to 100 μM CCh reached a maximum at about 10 s after the stimulation began, and hence the maximum response of the CCh (100 μM)-induced initial peak had already been attained before the concentration of CCh in the column was about 10 % of the applied CCh. Thus, the EC50 for CCh to evoke the initial peak would be much lower than the value mentioned above. Similar results were observed when lower concentrations of CCh (1 and 10 μM) were used (data not shown).

Role of extra- and intracellular Ca2+ on CCh-induced amylase secretion

In a Ca2+-free medium, in which extracellular Ca2+ was removed and 0.2 mM EGTA was added, the CCh (100 μM)-induced sustained plateau response of amylase secretion was almost abolished (Fig. 2A). Switching to a Ca2+-free medium during the CCh-induced sustained plateau led to a rapid return to near-resting levels. The CCh-induced sustained response was modestly increased by increasing the concentration of extracellular Ca2+ ([Ca2+]o) from 1 to 10 mM (Fig. 2B). On the other hand, the CCh-induced initial peak response changed little with [Ca2+]o when [Ca2+]o was changed simultaneously with CCh. Preperfusion in the Ca2+-free medium, however, decreased the initial peak response to CCh time dependently; the CCh (100 μM)-induced initial peak responses by the cells preperfused in the Ca2+-free solution for 2 and 5 min were 79.7 ± 12.0 and 44.3 ± 6.6 %, respectively, of the corresponding control solution (1.50 ± 0.14 % min−1, n = 3).

Figure 2. Effect of extracellular Ca2+ on CCh-induced amylase secretion.

Figure 2

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A, effect of CCh on amylase secretion in the presence (1 mM) and absence (Ca2+ omission plus 0.2 mM EGTA) of extracellular Ca2+. Extracellular Ca2+ was removed either simultaneously with the onset of, or 5 min after, CCh (100 μM) stimulation. B, effects of various concentrations of extracellular Ca2+ ([Ca2+]o) on the CCh (100 μM)-induced initial peak and sustained plateau. The sustained responses were calculated as means from 4 to 5 min after the onset of stimulation with CCh. [Ca2+]o was changed simultaneously with the onset of CCh stimulation. C, effect of NiCl2 on CCh-induced amylase secretion. NiCl2 (5 mM) was added either 2 min before, or 5 min after, CCh (100 μM) stimulation. Means are shown with their standard errors. Numbers in parentheses show the number of independent cell preparations.

NiCl2 has been reported to act as a Ca2+-entry blocker and to decrease the CCh-induced sustained plateau of [Ca2+]i to near-resting levels in rat parotid acinar cells (Merritt & Rink, 1987). When 5 mM NiCl2 was added 2 min before CCh, CCh (100 μM) produced only an initial transient peak (Fig. 2C). When NiCl2 was added during the CCh-induced sustained plateau, the rate of amylase secretion returned rapidly to near-resting levels. Similar results were observed when 1 mM CoCl2, another Ca2+-entry blocker, was used instead of NiCl2 (data not shown). Thus, the influx of Ca2+ into cells is crucial for the CCh-induced sustained plateau, but not for the initial peak response of amylase secretion.

Thapsigargin inhibits the microsomal Ca2+-ATPase re-uptake pump in rat parotid acinar cells (Takemura et al. 1989). As a consequence of this action, thapsigargin depletes Ca2+ in the InsP3-sensitive intracellular stores and increases [Ca2+]i in the rat parotid acinar cells (Foskett, 1991). Thapsigargin (1 μM) alone, however, only slightly increased the rate of amylase secretion, with its response being 0.10 ± 0.02 % min−1 (n = 6). The initial peak response induced by 100 μM CCh 5 min after continuous stimulation with 1 μM thapsigargin was 0.84 ± 0.10 % min−1, which was 55.7 ± 7.0 % of the corresponding control (1.52 ± 0.16 % min−1, n = 6) (Fig. 3A). However, preincubation with thapsigargin did not decrease the CCh-induced sustained plateau response. Preincubation for 5 min with 20 μM BAPTA AM, an intracellular Ca2+ chelator, which would reduce the contribution of InsP3-sensitive Ca2+ stores to [Ca2+]i, decreased the CCh-induced initial peak response significantly (P < 0.05); the magnitude of its response was 43.5 ± 12.2 % of the corresponding control (1.26 ± 0.14 % min−1, n = 3; Fig. 3A, right plot). Ionomycin, a calcium ionophore, which increases [Ca2+]i greatly, had only a slight effect on amylase secretion in the rat parotid acinar cells. In our perfusion system, ionomycin (2 μM) also evoked biphasic stimulation of amylase secretion with an initial rapid peak (0.62 ± 0.12 % min−1, n = 4) and a following sustained plateau (0.38 ± 0.04 % min−1). In the Ca2+-free medium, ionomycin (2 μM) evoked only an initial transient peak (0.66 ± 0.12 % min−1, n = 4). Preperfusion with 2 μM ionomycin for 5 min significantly decreased the magnitude of the CCh (100 μM)-induced initial peak response: 0.92 ± 0.22 % min−1 in the medium containing 1 mM Ca2+ and 0.24 ± 0.04 % min−1 in the Ca2+-free medium, 48.9 ± 6.0 and 12.3 ± 1.8 %, respectively, of the corresponding control (1.88 ± 0.26 % min−1, n = 4) obtained from the same cell preparations (Fig. 3B). Ionomycin, which increases the permeability of cellular membranes to Ca2+, would decrease Ca2+ in the InsP3-sensitive stores, the effect of which should be greater in a Ca2+-free medium. These results suggest that the CCh-induced initial rapid rise in [Ca2+]i, which is due to Ca2+ released from InsP3-sensitive intracellular stores, is crucial in evoking the CCh-induced initial peak of amylase secretion. The vehicle, ethanol (0.02 %), neither stimulated amylase secretion nor changed the effect of CCh on amylase secretion.

Figure 3. Effects of preperfusion with thapsigargin, BAPTA-AM and ionomycin on CCh-induced amylase secretion.

Figure 3

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Perfused parotid acinar cells were stimulated with 100 μM CCh 5 min after continuous stimulation with 1 μM thapsigargin (A, left plots), 20 μM BAPTA AM (A, right plots), or 2 μM ionomycin with or without 1 mM Ca2+ (B). Means of six (A, left), three (A, right), and four (B) paired experiments from independent cell preparations are shown with their standard errors.

Effect of PDBu on amylase secretion

Phorbol esters are believed to activate PKC by the same mechanism as DAG (Nishizuka, 1984). The effect of PDBu, an active phorbol ester, on amylase secretion developed slowly (Fig. 4A): a significant and maximum increase in amylase secretion with 1 μM PDBu was observed at about 90 s and 5 min, respectively, after the onset of stimulation. The PDBu-induced maximum rate of amylase secretion was 0.54 ± 0.04 % min−1 (n = 4), about three times the resting rate. An inactive control of PDBu, 4α-PDBu (1 μM), did not increase the rate of amylase secretion (data not shown). The vehicle, DMSO (0.02 %), changed neither the resting rate of amylase secretion nor the effect of CCh on amylase secretion.

Figure 4. Potentiation of amylase secretion between CCh and PDBu.

Figure 4

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Perfused parotid acinar cells were stimulated with either CCh or PDBu alone or with a combination of both. A, time profiles of amylase secretion induced by 1 μM PDBu, 100 μM CCh, or a combination of both. B, CCh (100 μM) was added either simultaneously with 1 μM PDBu or 2 and 5 min after continuous stimulation with 1 μM PDBu. C, summary of the concentration-responses of the CCh (100 μM)-induced initial peak and sustained plateau of amylase secretion 2 min after continuous stimulation with 1 μM PDBu. Means of three (A), six (B) and four (C) independent cell preparations are shown with their standard errors.

Potentiation of amylase secretion between CCh and PDBu

A combination of CCh (100 μM) and PDBu (1 μM) when added simultaneously evoked biphasic stimulation of amylase secretion with an initial peak (2.52 ± 0.18 % min−1, n = 3) and a following sustained plateau (1.30 ± 0.06 % min−1; Fig. 4A). The magnitude of both responses was significantly (P < 0.05 for both responses) higher than the sum of the responses induced by each agonist alone. The CCh-induced initial peak response, but not the sustained plateau, was further enhanced by prior perfusion with PDBu in a time-dependent manner (Fig. 4B). Thus, the CCh (100 μM)-induced initial peaks 2 and 5 min after continuous stimulation with 1 μM PDBu were 2.96 ± 0.18 % min−1 and 3.40 ± 0.28 % min−1, respectively. Further increase in the time of preincubation with 1 μM PDBu up to 10 min did not enhance the CCh-induced initial peak response (data not shown). The time course of changes in the initial peak of amylase secretion induced by the combination of CCh and PDBu was very similar to that induced by CCh alone.

The concentration-responses of CCh-induced amylase secretion 2 min after continuous stimulation with 1 μM PDBu are shown in Fig. 4C. The maximum response and the EC50 for CCh to evoke the initial peak were 100 μM and about 3 μM, respectively, which were not different from those induced by CCh alone. (The maximum response and the EC50 for CCh alone were 100 μM and 5 μM, respectively. See Fig. 1.) The sustained plateau response reached a maximum at 1 μM CCh, which was lower than that induced by CCh alone (10 μM, in Fig. 1). These results suggest that PDBu increases the sensitivity for CCh to evoke the sustained response, but not the initial peak.

When the order of stimulation with CCh and PDBu was reversed, that is, PDBu was added 5 min after continuous stimulation with CCh, the PDBu (1 μM)-induced increase in amylase secretion was much higher than that induced by PDBu alone (Fig. 5). Furthermore, the effect of PDBu was dependent on the concentration of CCh and the PDBu effect was maximum at 1 μM CCh. In addition, the PDBu-induced increase in amylase secretion in the presence of 1 μM CCh developed quite rapidly and reached a maximum at about 90 s after stimulation, with this value being 0.82 ± 0.08 % min−1 (n = 5). When a higher concentration of CCh (10 or 100 μM) was used instead of 1 μM CCh, the effect of PDBu developed slowly and its maximum effect was lower; the PDBu (1 μM)-induced increase in amylase secretion, which reached a maximum at about 5 min, was 0.60 ± 0.10 % min−1 (n = 5) for 100 μM CCh.

Figure 5. PDBu-induced increase of amylase secretion during stimulation with CCh.

Figure 5

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Parotid acinar cells were first stimulated with various concentrations of CCh. Then the PDBu (1 μM)-induced increase in amylase secretion was examined 5 min after continuous stimulation with CCh. Means are shown with their standard errors. Numbers in parentheses show the number of independent cell preparations.

When stimulation with CCh and PDBu in combination was terminated by switching to a medium without agonists, the rate of amylase secretion declined rapidly. The time required to return to half of the initial level (T½), was about 90 s for 100 μM CCh and 40 s for 1 μM CCh; these values were similar to those obtained by using CCh alone. The decline in PDBu-induced amylase secretion, on the other hand, developed quite slowly and T½ was more than 5 min.

The effect of PDBu on the potentiation was concentration dependent; the minimum and maximum effects of PDBu were obtained at 10 nM and at 1 μM, respectively (data not shown). Similar potentiation of amylase secretion was observed when another active phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 μM), was used instead of PDBu (data not shown). 4α-PDBu (1 μM), when added 5 min after continuous stimulation with CCh (1 μM), increased amylase secretion slightly, but its effect was only about 10 % of that induced by 1 μM PDBu.

Figure 6 shows the effects of extracellular Ca2+ on amylase secretion induced by PDBu or CCh alone and by CCh and PDBu in combination. Amylase secretion induced by PDBu (1 μM) alone in the Ca2+-free medium was 0.26 ± 0.02 % min−1 (n = 7), which was lower than the corresponding control in the presence of 1 mM Ca2+ (0.65 ± 0.08 % min−1, n = 5) obtained with the same cell preparations. Switching to a medium containing 1 mM Ca2+ at 5 min after continuous stimulation with 1 μM PDBu in the Ca2+-free medium produced only a small and sustained response of amylase secretion (0.16 ± 0.04 % min−1, n = 3). When the CCh (100 μM) effect was examined at 5 min after continuous stimulation with 1 μM PDBu in the Ca2+-free medium, CCh evoked only an initial transient peak and did not produce a sustained plateau response. The CCh-induced initial peak of amylase secretion in the Ca2+-free medium was 2.98 ± 0.12 % min−1 (n = 5) which was not significantly different from the corresponding control (3.38 ± 0.12 % min−1) obtained with the same cell preparations. The PDBu (1 μM)-induced increase of amylase secretion at 5 min after continuous stimulation with 100 μM CCh in the Ca2+-free medium was 0.26 ± 0.04 % min−1, which was significantly (P < 0.01) lower than that of the corresponding control (1.16 ± 0.08 % min−1, n = 5).

Figure 6. Ca2+ dependency of amylase secretion induced by CCh, PDBu or a combination of both.

Figure 6

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Parotid acinar cells were first stimulated with 1 μM PDBu or 100 μM CCh in a Ca2+-free medium. Five minutes after continuous stimulation with each agonist, the reverse was used as a stimulant. Means of five independent cell preparations are shown with their standard errors.

Switching to a Ca2+-containing solution after continuous stimulation with CCh (100 μM) in the Ca2+-free medium again evoked a second biphasic response of amylase secretion with a rapid and large, but transient peak and a following sustained plateau (Fig. 7, left plot). The magnitude of the second peak was dependent on the time of preperfusion in the Ca2+-free medium; 3 min of perfusion without Ca2+ was sufficient to obtain the maximum response of the second peak (data not shown). The Ca2+ (1 mM)-induced second peak, which reached a maximum at about 90 s after the onset of stimulation, was about 60 % of the CCh-induced initial peak. A higher concentration of Ca2+ (5 mM) enhanced the magnitude of the second response in both the initial peak and the sustained plateau (Fig. 7, right plot). It also slightly reduced the time for Ca2+ to evoke the second peak. A more precise time resolution of the Ca2+ effect using Lucifer Yellow demonstrated that the responses to 1 and 5 mM Ca2+ reached a maximum at about 60 and 30 s, respectively, after the start of stimulation (data not shown). PDBu (1 μM) also increased the magnitude of the Ca2+-induced second peak but only marginally reduced the time for Ca2+ to elicit the maximum response (about 50 s), suggesting that PDBu did not increase the sensitivity for Ca2+ to evoke a rapid effect. This is consistent with the results showing that PDBu did not decrease the EC50 for CCh to evoke the initial peak response (Fig. 4).

Figure 7. Effect of switching from Ca2+-free to Ca2+ (1 or 5 mM)-containing media during the CCh-induced sustained plateau.

Figure 7

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Parotid acinar cells were first stimulated with 100 μM CCh (left and right plots) or with a combination of 100 μM CCh and 1 μM PDBu (middle plot) in a Ca2+-free medium for 5 min, and then perfusion solutions were switched to a medium containing 1 mM (left and middle plots) or 5 mM (right plot) Ca2+. Means are shown with their standard errors. Numbers in parentheses show the number of independent cell preparations.

Effect of PKC inhibitors on amylase secretion induced by CCh, PDBu and a combination of both

Ro 31–8220 has been described as a specific and powerful inhibitor of PKC (Murphy & Westwick, 1992). Ro 31–8220 (10 μM), however, did not significantly decrease amylase secretion induced by CCh (100 μM) alone in either the initial peak or the sustained plateau responses (Fig. 8A). Also, Ro 31–8220 only slightly decreased amylase secretion induced by PDBu. The PDBu (1 μM)-induced rises in amylase secretion in the absence and presence of 10 μM Ro 31–8220 were 0.38 ± 0.04 and 0.34 ± 0.06 % min−1 (n = 3), respectively (Fig. 8B). Ro 31–8220, however, markedly decreased amylase secretion induced by a combination of CCh and PDBu whichever was added first. The PDBu (1 μM)-induced increment of amylase secretion at 5 min after continuous stimulation with 100 μM CCh in the presence of Ro 31–8220 (10 μM) was 0.10 ± 0.02 % min−1, which was significantly (P < 0.01) lower than that of the corresponding control (0.82 ± 0.10 % min−1, n = 3) (Fig. 8A). In the presence of Ro 31–8220, CCh evoked only an initial transient peak when its effect was examined at 5 min after continuous stimulation with 1 μM PDBu, but it did not produce a sustained plateau response; the CCh (100 μM)-induced initial peak response was 1.94 ± 0.14 % min−1, which was significantly (P < 0.01) lower than the corresponding control (3.56 ± 0.38 % min−1, n = 3; Fig. 8B). Ro 31–8220, when added during the sustained plateau phase induced by CCh and PDBu in combination, rapidly decreased the rate of amylase secretion (data not shown). Thus, Ro 31–8220 markedly decreased amylase secretion induced by PDBu and CCh in combination but only slightly decreased the effect induced by CCh or PDBu alone. Essentially similar results were also obtained with another inhibitor of PKC, staurosporine.

Figure 8. Effect of Ro 31–8220 on amylase secretion induced by CCh or PDBu alone or a combination of both.

Figure 8

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Perfused parotid acinar cells were stimulated with 100 μM CCh (A) first or 1 μM PDBu (B) first in the presence and absence of 10 μM Ro 31–8220. Five minutes after continuous stimulation with one of the agonists, the other was used as the stimulant. Ro 31–8220 was added 2 min before the onset of stimulation with each agonist. Means of three (A and B) paired experiments from independent cell preparations are shown with their standard errors.

Effect of CCh on [Ca2+]i

CCh evoked a characteristic biphasic change in [Ca2+]i with an initial peak followed by a gradual decline leading to almost a plateau at about 3 min. The CCh-induced increases of the initial peak and the sustained plateau at 3 min after stimulation were 461 ± 65 and 235 ± 27 nM (n = 9), respectively, in the medium containing 1 mM Ca2+. In Ca2+-free medium (Ca2+ omission plus 0.2 mM EGTA), CCh evoked only an initial transient peak (251 ± 16 nM, n = 4). The concentration-response of [Ca2+]i induced by CCh shows that the EC50 of the CCh-induced initial peak and sustained plateau responses were about 2 μM and 0.3 μM, respectively. PDBu (1 μM) alone did not increase [Ca2+]i, and preincubation with 1 μM PDBu for 2 min did not enhance either the CCh (100 μM)-induced initial peak or the sustained plateau of [Ca2+]i (data not shown).

DISCUSSION

Ca2+ is crucial in amylase secretion induced by CCh

Column perfusion of parotid acinar cells is very useful for studying the dynamic changes of amylase secretion evoked by various agonists (Yoshimura & Nezu, 1991, 1992). In the present experiments, we applied this system for examining the effect of continuous stimulation with CCh on amylase secretion under various experimental conditions in a more precise time resolution and obtained the following results suggesting the importance of Ca2+ in CCh-induced amylase secretion. (1) The time course of CCh-induced amylase secretion was similar to that of the CCh-induced [Ca2+]i response. Thus, CCh-induced amylase secretion developed almost simultaneously with the onset of CCh stimulation and reached a maximum at about 10 s. These results were quite different from that of the CCh-induced amylase secretion in perfused pancreatic acinar cells which has been reported to be a relatively slow process (Tsunoda et al. 1990). (2) Comparison of the concentration-response of CCh-induced amylase secretion and [Ca2+]i showed that both parameters changed in parallel. (3) The CCh-induced sustained plateau of amylase secretion was almost abolished when the influx of Ca2+ into cells was inhibited, these conditions blocking the CCh-induced sustained [Ca2+]i response. The CCh-induced initial peak, on the other hand, was little decreased when extracellular Ca2+ was removed at the time of stimulation with CCh. (4) The CCh-induced initial peak response was greatly decreased by preincubation in a Ca2+-free medium time dependently and in a medium with thapsigargin or ionomycin, which would decrease Ca2+ in InsP3-sensitive stores. Time-dependent decrease by preincubation in a Ca2+-free medium has been reported by Nauntofte & Dissing (1987) in a CCh-induced initial peak of [Ca2+]I response. (5) Restoration of extracellular Ca2+ after a CCh-induced transient amylase response in a Ca2+-free medium, which evokes a rapid increase in [Ca2+]i, elicited a second rapid and large peak of amylase secretion. Our results also suggest that Ca2+ released from the InsP3-sensitive stores is crucial for the CCh-induced initial peak response of amylase secretion, whereas the sustained plateau response was dependent on Ca2+ entry into cells. A similar dependency of CCh-induced amylase secretion on intra- and extracellular Ca2+ has been reported in pancreatic acinar cells (Tsunoda et al. 1990). 2,4-Dinitrophenol (2,4-DNP) and oligomycin, which decrease cellular ATP, abolished the CCh-induced sustained response of amylase secretion but only slightly decreased the initial peak (data not shown). The concentration of CCh needed to evoke a sustained plateau was about ten times lower than that needed to produce an initial peak. Together with the difference in the Ca2+ dependency, the mechanism for evoking the CCh-induced initial peak was considered to be different from that of the sustained response.

One major problem in postulating a crucial role for Ca2+ in CCh-induced amylase secretion is that ionomycin and thapsigargin, which greatly increase [Ca2+]i in rat parotid acinar cells (Foskett et al. 1991; Tojyo et al. 1992), have only a very limited effect in stimulating amylase secretion (Fig. 3). A Ca2+ signal upon stimulation with CCh has been reported to be initiated at a localized pole in parotid acinar cells (Dissing et al. 1990; Liu et al. 1998) and in other exocrine cells (Lee et al. 1997). When we measured CCh-induced [Ca2+]i changes in single cells or cell suspensions, a greater, but localized [Ca2+]i signal was obscured by lower, but more diffused [Ca2+]i changes in the rest of the cells. Ionomycin, which non-specifically increases permeability of cellular membranes to Ca2+, would evoke a more diffused rise in [Ca2+]i. Thus, the ionomycin-induced localized rise in [Ca2+]i transients, which is crucial for the regulation of amylase secretion, may be much lower than that induced by CCh. In accord with this perspective, near-membrane [Ca2+]i transients recorded by using the calcium indicator FFP18 has recently been reported to reach micromolar levels when bulk cytoplasmic [Ca2+]i changes recorded by using fura-2 rose to only a few hundred nanomolar (Etter et al. 1996). In addition, it has been reported that a very high concentration of [Ca2+]i (about 30 μM), which is about 50-fold higher than the CCh-induced maximum [Ca2+]i response observed with fura-2, is required for eliciting the maximum response of amylase release in permeabilized pancreatic acinar cells (Stecher et al. 1992; O'Sullivan & Jamieson, 1992).

The rise in [Ca2+]i induced by thapsigargin has been reported to develop very slowly; it takes more than 5 min for thapsigargin to attain its maximum effect on [Ca2+]i (Foskett et al. 1991). Jonas et al. (1994) reported the development of desensitization to Ca2+-induced insulin secretion in α-toxin-permeabilized HIT-T15 cells. Thus, one possibility for explaining the very slight effect of thapsigargin on amylase secretion is that the thapsigargin-induced gradual rise in [Ca2+]i simultaneously develops rapid desensitization to the effect of Ca2+. It does appear, however, that our results cannot be explained by this postulate, since application of a linear gradient of Ca2+ to 1 mM over 5 min after continuous stimulation with CCh in a Ca2+-free medium only slightly decreased the magnitude of the Ca2+-induced second peak of amylase secretion (data not shown).

Activation of PKC is not required for CCh-induced amylase secretion

Ca2+-mobilizing agonists, in addition to their effects on [Ca2+]i, activate PKC by increasing DAG (Nishizuka, 1984). Machado-De Domenech & Söling (1987) reported that stimulation with CCh was accompanied by the translocation of PKC activity from the cytosol to the particulate fractions in guinea-pig parotid acinar cells. Möller et al. (1996) showed that downregulation of PKC by about 90 % after treatment with PMA does not decrease CCh-induced amylase secretion, but the PKC inhibitor, Ro 31–8220 abolishes the CCh-induced amylase secretion. They suggested that muscarinic stimulation of secretion involves the activity of a PKC isozyme that is resistant to downregulation by PMA in guinea-pig parotid acinar cells. In rat parotid slices, we observed the translocation of PKC activity from the cytosolic to the membrane fractions with PDBu, but we were not able to observe a significant translocation of PKC with CCh (data not shown). In the present experiments, PKC inhibitors (Ro 31–8220 and staurosporine) did not significantly decrease CCh-induced amylase secretion. Furthermore, activation of PKC by PDBu did not simulate the time course of CCh-induced amylase secretion. In addition, activation of PKC by PDBu largely potentiated the CCh-induced amylase secretion. These results suggest that PKC contributes little to CCh-induced amylase secretion in rat parotid acinar cells. Thus, there may be some species differences in the role of PKC in CCh-induced amylase secretion. CCh stimulates phospholipase C, thereby increasing DAG, but DAG produced by CCh may not be sufficient to activate the PKC isozyme which is necessary to enhance Ca2+-stimulated amylase secretion. Similar results indicating that the activation of PKC is not required for Ca2+-dependent exocytosis have been reported in permeabilized chromaffin cells (Terbush & Holz, 1990) and in permeabilized pancreatic acinar cells (O'Sullivan & Jamieson, 1992).

Mechanism of the potentiation of amylase secretion between CCh and PDBu

The initial rise in amylase secretion induced by CCh and PDBu in combination was very similar to that induced by CCh but not by PDBu. The initial decline in the rate of amylase secretion induced by CCh and PDBu in combination, when the perfusion solution was switched to a medium without the agonists, developed more rapidly than that induced by PDBu alone. These results suggest that potentiation of amylase secretion between CCh and PDBu is mainly evoked by a PDBu-induced modification of the CCh effect, not by a CCh-induced modification of the PDBu effect.

The following evidence suggests that PDBu potentiates CCh-induced amylase secretion by activating PKC. (1) The concentration of PDBu needed to evoke the potentiation of amylase secretion was similar to that needed for activating PKC (translocation of PKC from cytosolic to membrane fractions; data not shown). (2) The effect of PDBu on the potentiation was stereospecific (4α-PDBu, a negative control of phorbol ester, only slightly potentiated the CCh-induced amylase secretion). (3) The effect of PDBu on the potentiation was markedly decreased by inhibitors of PKC.

In our results with perfused parotid acinar cells, CCh mainly regulated amylase secretion by changes in [Ca2+]i. PDBu, however, neither increased [Ca2+]i nor enhanced the effect of CCh on [Ca2+]i (data not shown), and hence the potentiation of CCh-induced amylase secretion by PDBu was not explained by changes in [Ca2+]i. Thus, our results suggest that the potentiating effect of PDBu on CCh-induced amylase secretion is due to a PDBu-induced modification of the Ca2+ effect. However, we only measured [Ca2+]i with cell suspensions or single cells and it is therefore possible that PDBu enhanced the CCh-induced localized [Ca2+]i transients, the effect of which was not detected by our [Ca2+]i measurements.

Concentration-response analysis of amylase secretion induced by CCh with and without PDBu shows that PDBu enhanced the magnitude of CCh-induced amylase secretion in both the initial peak and sustained plateau responses. PDBu, however, did not increase the sensitivity of CCh to evoke the initial peak. PDBu also did not increase the Ca2+ sensitivity of amylase secretion to restoring extracellular Ca2+ (Fig. 8). These results suggest that PDBu did not increase the Ca2+ sensitivity for evoking rapid response of amylase secretion. PDBu, on the other hand, modestly increased the sensitivity of the CCh-induced sustained response. Our results thus again suggest that the mechanism for CCh and/or Ca2+ to evoke the rapid response of amylase secretion differs from that of the sustained response. In permeabilized parotid acinar cells, Rubin & Adolf (1994) showed that dioctanoylglycerol, a DAG analogue, increases the Ca2+ sensitivity, but not the amplitude, of amylase secretion. In our preliminary results with α-toxin-permeabilized parotid acinar cells, however, PDBu enhanced the efficacy for Ca2+ to stimulate amylase secretion in both the initial rise and a later effect (data not shown) which was consistent with our results showing that PDBu enhanced the efficacy of the effects of CCh and Ca2+ in intact parotid acinar cells.

Possible scheme of amylase secretion induced by CCh, PDBu and the combination of CCh and PDBu

In permeabilized chromaffin cells (Holz et al. 1989), PC12 cells (Hay & Martin, 1992) and insulinoma cells (Efanov et al. 1997), exocytosis has been assumed to be composed of at least two distinct steps; one is the ATP-dependent but Ca2+-independent priming of inactive granules, and the other is a final fusion/secretion of the primed granules in response to a rise in [Ca2+]i which is relatively ATP independent. A similar model for the regulation of exocytosis has recently been reported for the exocrine cells such as permeabilized pancreatic acini (Padfield & Panesar, 1997) and intact parotid acinar cells (Segawa, 1997; Segawa & Yamashina, 1998; Yoshimura et al. 1998). Furthermore, activation of PKC has been suggested to promote the priming of secretory granules for exocytosis in chromaffin cells (Burgoyne, 1991), PC12 cells (Hay & Martin, 1992) and insulinoma cells (Efanov et al. 1997) or to increase the size of readily releasable pools of secretory granules in adrenal chromaffin cells (Gillis et al. 1996).

In rat parotid acinar cells (Fig. 9), CCh activates phospholipase C to produce InsP3 and DAG. An InsP3-induced rapid and large rise in [Ca2+]i triggers final fusion/exocytosis of the primed granules, thereby evoking an initial rapid and large, but transient peak of amylase secretion. The CCh-induced sustained plateau response of amylase secretion would probably represent the balance of two independent processes; priming of secretory granules and Ca2+-induced final fusion/exocytosis. In the absence of extracellular Ca2+, the CCh-induced rapid and large [Ca2+]i transient produces only an initial transient peak of amylase secretion. CCh in a Ca2+-free medium however, would have some intrinsic ability to promote the priming of secretory granules, thereby accumulating the primed granules time dependently. Thus, rapid influx of Ca2+ into cells by restoring extracellular Ca2+ after continuous stimulation with CCh in a Ca2+-free medium evokes a second large peak of amylase secretion. The priming of secretory granules induced by CCh is not mediated by PKC, since the PKC inhibitor Ro 31–8220 was not effective in inhibiting the effect of CCh. Activation of PKC by PDBu also promotes the priming of secretory granules, thereby enhancing the efficacy, but not potency, of Ca2+ to trigger the final fusion/exocytosis. A CCh-induced increase in DAG, if any, would not be sufficient for activating PKC. Our results of the effect of 2,4-DNP on CCh-induced amylase secretion are consistent with this model.

Figure 9. Possible scheme illustrating the roles of Ca2+ and PKC in amylase secretion induced by CCh and PDBu.

Figure 9

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There are at least two forms of secretory granules in the parotid acinar cells; one is not activated for fusion, and the other is activated granules ready for the final fusion/exocytosis. Thus, the exocytotic process of amylase secretion in parotid acinar cells can be separated into at least two biochemically distinct steps; the first step is ATP-dependent priming of secretory granules, and the second step is Ca2+-activated fusion of the primed granules. CCh activates phospholipase C (PLC), thereby increasing InsP3 and DAG. InsP3, by releasing Ca2+ from InsP3-sensitive intracellular stores, increases [Ca2+]i, which stimulates the final fusion/exocytosis. The accumulation of DAG by CCh, if any, would not be sufficient to activate PKC, and hence to enhance the Ca2+-evoked final fusion/exocytosis itself. CCh alone also has some intrinsic, but unknown, mechanism for promoting the priming of secretory granules. Activation of PKC by PDBu stimulates the priming, thereby increasing the efficacy of Ca2+ to stimulate amylase secretion. PDBu also slightly stimulates the priming of secretory granules which is independent of the activation of PKC.

We previously reported that cAMP promotes the priming of secretory granules, thereby enhancing Ca2+-induced triggering of the final fusion/secretion in rat parotid acinar cells (Yoshimura et al. 1998). Our present results also suggested that PDBu, by activating PKC, enhanced the Ca2+-induced amylase secretion by increasing the priming of the secretory granules. Our previous results that show additive stimulation of amylase secretion between isoproterenol and PDBu are consistent with this model (Yoshimura et al. 1998). In addition, CCh has been suggested to have some intrinsic ability to prime the secretory granules. In our previous study, cAMP potentiated the Ca2+-induced amylase secretion by greatly increasing both the efficacy and the potency of the Ca2+ effect. On the other hand, PDBu increased the CCh-induced maximum response but did not increase the sensitivity for Ca2+ to evoke a rapid response of amylase secretion. These results suggest that the mechanism of priming induced by the PKC-messenger system and/or by CCh alone may be different from that induced by the cAMP-messenger system. This difference in the mechanism of priming may partly explain the results showing that amylase secretion induced by the cAMP messenger, but not by CCh alone or CCh and PDBu in combination, only slightly decreased in a Ca2+-free medium.

In conclusion our results show that the rise in [Ca2+]i induced by CCh is crucial for the final fusion/exocytosis of amylase secretion in rat parotid acinar cells. CCh also has some ability to promote the priming of secretory granules, but its effect is not mediated by activation of PKC. PDBu, by activating PKC, potentiates CCh-induced amylase secretion by increasing the priming of secretory granules, thereby enhancing the efficacy of Ca2+ to trigger final fusion/exocytosis.

Acknowledgments

This work was supported in part by a grant-in-aid from the Akiyama Foundation, Japan. We also acknowledge Dr Yukiharu Hiramatsu for his measurements of the effect of PDBu on the CCh-induced [Ca2+]i response.

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