stimulated mucin secretion was partially inhibited by removal of extracellular Ca2+ [5,6], and isoprenaline increased 46Ca2+ efflux from rat submandibular acini ...
Biochem. J. Biochem.
J.
(1993) 293, 691-695 (Printed in Great Britain) (1993)
293,
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691-695
fl-Adrenergic mobilization of Ca2+ from submandibular acini
691
an
intracellular store in rat
Chris LLOYD MILLS,* Maurice B. HALLETT,t Margaret A. MCPHERSON*: and Robert L. DORMER* *Department of Medical Biochemistry and tDepartment of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
Increases in cytoplasmic free Ca2l concentration in rat submandibular acini were observed in response to isoprenaline (10 ,uM), noradrenaline (10 #uM) and carbamoylcholine (10 ,M). Noradrenaline and carbamoylcholine responses were decreased to 27% and 33 % respectively in the absence of extracellular
Ca2l, suggesting a major requirement for Ca2l entry. /6Adrenergic stimulation elicited a small (35-40 nM) free Ca2+ rise, approx. 75 % of which was mobilized from an intracellular store. Results suggest that this Ca2+ rise is a key event in the physiological triggering of mucin secretion by exocytosis.
INTRODUCTION
MATERIALS AND METHODS Materials Soya-bean trypsin inhibitor (type 1-S), hyaluronidase (type 1), atropine sulphate, carbamoylcholine chloride, (-)-isoprenaline hydrochloride, (-)-noradrenaline hydrochloride and DL-propranolol were purchased from Sigma. Phentolamine mesylate [2N-(3-hydroxyphenyl)-p-toluidomethyl-2-imidazoline mesylate; Rogitine] was from CIBA. Collagenase was obtained from
Secretion from salivary glands is controlled by both sympathetic and parasympathetic nerves. Salivary acinar cells thus present a good cell model to investigate second-messenger responses to cholinergic and adrenergic receptor occupancy in regulating exocytosis and Cl- secretion [1,2]. In addition, they have been used for investigating abnormalities in cystic fibrosis cells, which show defective ,-adrenergic stimulation of protein and Clsecretion [3]. Mucin secretion from rat submandibular acinar cells is primarily mediated by stimulation of ,6-adrenergic receptors [4-6], cholinergic agonists being much less potent [2]. The secretagogue activity of forskolin, cyclic nucleotide phosphodiesterase inhibitors and synthetic cyclic AMP analogues suggests that cyclic AMP can act as a trigger of exocytosis [7,8]. Increased cellular cyclic AMP levels, leading to activation of protein kinase A and phosphorylation of specific proteins [8,9], have been shown to result from ,-adrenergic stimulation of submandibular acinar cells. However, incorporating excess cyclic AMP phosphodiesterase into intact acinar cells, by a hypotonic-swelling method [1], abolished the cyclic AMP rise elicited by a maximal concentration of isoprenaline, without affecting stimulation of mucin secretion. Indirect evidence has indicated that Ca2l is involved in fi-adrenergic stimulation of mucin secretion. Thus isoprenalinestimulated mucin secretion was partially inhibited by removal of extracellular Ca2+ [5,6], and isoprenaline increased 46Ca2+ efflux from rat submandibular acini [6]. Furthermore, introduction of the Ca2+ chelator BAPTA [bis-(o-aminophenoxy)ethaneNNN'N'-tetra-acetic acid] into submandibular acini inhibited secretion in response to fi-adrenergic and cholinergic agonists [2]. It was therefore suggested that Ca2+ is necessary for ,-adrenergic stimulation of secretion and acts at a common distal point in the pathway of stimulation of exocytosis. In order to investigate the effects of physiological stimulation on Ca2+ homoeostasis and whether Ca2+ is released from an intracellular store, we have examined the actions of adrenergic and cholinergic agonists on cytoplasmic free Ca2+ concentrations in isolated rat submandibular acini, as measured by the fluorescent indicator fura-2. The data suggest that limited Ca2+ release from an Ins(1,4,5)P3-insensitive store is involved in stimulating mucin secretion by exocytosis.
Cooper Biomedical, minimal Eagle's medium amino acid supplement and Hanks' balanced salt solution were from Gibco, and BSA fraction V was from ICN. Fura-2 acetoxymethyl ester (fura-2 AM) was from Molecular Probes, Eugene, OR, U.S.A. All other reagents were from BDH, and were of the highest purity available.
Preparation of submandibular acini and measurement of mucin secretlon Submandibular acini were prepared from overnight-starved male Wistar rats (250-300 g), as described previously [2,6]. Briefly, tissue was cut into small fragments after removal of major ducts and incubated in Hanks' balanced salt solution containing 64 units/ml collagenase and 500 units/ml hyaluronidase for 60 min at 37 °C with shaking at 120 cycles/min. The tissue was dispersed by pipetting through a 2.4 mm-diameter polypropylene tip, and acini were purified by layering over Krebs-Henseleit bicarbonate buffer (KHB) [6] containing 4% BSA, and centrifuging for 4 min at 50 g. Purified acini were resuspended in KHB containing 2 % BSA. Mucin release was measured as previously described [2,6], after pulse-chase labelling with D-[13H]glucosamine hydrochloride. After incubation under experimental conditions at 37 °C, 3H-labelled mucins released into the medium at zero time and after 30 min were acid-precipitated and their radioactivity was measured as previously described [2,6]. Protein content of cell pellets was determined by the method of Lowry et al. [10], and mucin release over 30 min was expressed as d.p.m./mg of protein. The data are presented as percentages of basal secretion, to take account of variation in unstimulated mucin release between experiments.
Abbreviation used: KHB, Krebs-Henseleit bicarbonate buffer; BAPTA, bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid. $ To whom correspondence should be addressed.
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Loading of acini with fura-2 and measurement of cytoplasmic
Ca2+
Fura-2 was incorporated into acini by addition of fura-2 AM (1 #uM) dissolved in dimethyl sulphoxide to acini suspensions (approx. 106 acini/ml) in KHB containing 20% BSA. After 30 min at room temperature (approx. 20 °C), acini were washed by centrifugation (3 min at 30 g) before transfer to fresh KHB containing 2 % BSA. Fluorescence (505 nm) excited at 340 nm and 380 nm was recorded as described previously [1 1] by using a Spex Fluorolog II (Glen Creston, Stanmore, U.K.) with two lamps and monochromators, together with a mirrored optical chopper system. Autofluorescence of acini was less than 5 % of signal fluorescence. The 340 nm/380 nm ratio was calculated. At the end of each experiment, cells were lysed with 50 ,#M digitonin to obtain Rmax, and 50 mM EGTA added to the lysed cells to obtain Rmin. The intracellular Ca2+ concentration was calculated from the equation [Ca2+]1 = K (R - Rmin.)/(Rmax - R), where R is the ratio of 340 nm/380 nm fluorescence and K is 268.5 nM [1 1]. In order to ensure that changes in fura-2 fluorescence in response to agonists were intracellular and not due to release of fura-2 into the medium, it was shown that after loading and washing acini: (a) the spectrum of fura-2 corresponded to that of the free acid; (b) all fura-2 fluorescence sedimented with the acini when the cuvette stirrer was switched off. In addition, in certain experiments (see Figure 2), specific blockers were added during the course of the response to an agonist to show that the rise in Ca2+ concentration returned to the basal concentration and therefore could not be due to increased leakage or secretion of fura-2. Figures 1, 3 and 4 show data as a change in cytoplasmic free Ca2+, in order to present a direct comparison of responses in the absence or presence of extracellular Ca2+. Absolute free Ca2+ concentrations are shown in Table 1.
RESULTS Responses to agonists Isoprenaline (10 1tM), which is maximally effective for stimulation of mucin secretion [6], evoked a small sustained rise in cytoplasmic free Ca2+ to approx. 40 nM above resting. The response was inhibited in the presence of propranolol (30 ,uM) (Figure 1). The physiological neurotransmitter noradrenaline (10 ,uM) gave a greater increase in free Ca2+ (approx. 150 nM) which returned, within 20 s, to a plateau of sustained elevation, 60-70 nM above resting (Figure 2). The f-blocker propranolol (30 ,M), added during the plateau phase, lowered the Ca2+ concentration by approx. 40 nM; Ca2+ concentration was returned to resting by the subsequent addition of the oc-blocker phentolamine (30 ,uM). Phentolamine added at the same time as noradrenaline produced a response similar to that evoked by isoprenaline (compare Figures 1 and 2). This response was abolished by subsequent addition of propranolol (30 ,uM). The cholinergic agonist carbamoylcholine gave a rise in free Ca2+ concentration intermediate between that elicited by isoprenaline and noradrenaline, which was blocked by atropine (Figure 3). The time taken to reach the maximum free Ca2+ concentration was approx. 6-8 s for all agonists (Table 1). Table 1 shows combined data from several experiments. In terms of the maximally stimulated rise in cytoplasmic free Ca2+
concentration, the order of efficacy of agonists was: noradrenaline > carbamoylcholine > isoprenaline(= noradrenaline +phentolamine). This does not correlate with the order of efficacy of agonists in maximally stimulating mucin secretion, which was isoprenaline (10 1M) = noradrenaline (10 lM) > carbamoylcholine (10 ,uM) [2,6]. Nevertheless, isoprenaline
l~ ~
stimulation of mucin secretion [6] and intracellular Ca2+ concentration (Figure 1) were inhibited by propranolol.
Ca2+ mobilization
In order to determine whether agonists released Ca2+ from an internal store, cytoplasmic free Ca2+ concentrations were measured in Ca2+-free medium (cells were centrifuged and resuspended in KHB without added Ca2+ and containing 0.1 mM EGTA, 1-2 min before agonist addition). The response to isoprenaline was decreased by approx. 250% in the absence of
260 -S
-
Isoprenaline
N
U
14
40
Eco
020 -
0 0
1.'r0 -
C (U
0
0
50
25
75
Time (s)
Figure 1 lsoprenaline Increases cytoplasmic tree Ca2+
Cytoplasmic free Ca2+ concentration was measured in response to 10 ,FM isoprenaline in the ) or absence (----) of extracellular Ca2+ (Ca2+-free KHB containing 0.1 mM presence ( EGTA). Propanolol (30,uM) was added 30 s before isoprenaline addition in Ca2+-containing . The traces are representative of at least four experiments. Data are presented KHB ( ......). as change in free Ca2+ concentration to take account of the lower resting level in Ca2+-free medium (see Table 1).
300 c -S
250 -
u
.oco 200 E
'U 0
150
-
100
0
50
100
150
Time (s)
Figure 2 Effect of a- and fJ-adrenergic blockers on noradrenaline-induced changes In cytoplasmic tree Ca2+ Cytoplasmic free Ca2+ concentration was measured in response to 10 ,M noradrenaline ) or 10 1uM noradrenaline in the presence of 30 ,uM phentolamine ( . ), which was added 30 s before addition of noradrenaline. Subsequent addition of propranolol (30 ,uM) or phentolamine (30 ,uM) was as indicated.----, Resting cytoplasmic free Ca2+ concentration. Traces are representative of three experiments.
O
,-Adrenergic mobilization of Ca2+ from an intracellular store
693
200 -
150
2
+- 150-
.i 100-
*E100co
E
0. 0 5 50 m
0
C._
0
CD q-
CD_
C-
0
Figure 3 Carbamoylcholine Increases cytoplasmic free Ca2+ predominantly by Ca2+ Influx Cytoplasmic free Ca2+ concentration was measured in response to 10 uM carbamoylcholine in KHB ( ), Ca2+-free (+ 0.1 mM EGTA) KHB ( . ) or KHB containing 30 #uM atropine (----). Atropine was added 30 s before addition of carbamoylcholine. Traces are representative of at least four experiments.
Table 1 Cytoplasmic free Ca2+ changes In rat submandibular acini Data shows resting and peak free Ca2+ concentrations in response to agonists in the absence or presence of extracellular calcium (Ca2+-free KHB containing 0.1 mM EGTA). Results are means+ S.E.M. for the number of experiments shown in parentheses: ***P < 0.001, **P < 0.01, *P < 0.05 for difference from testing concentration, tP < 0.05 for difference from resting concentration in Ca2+-containing medium. Differences were assessed by Student's ttest. Abbreviations: cAMP, cyclic AMP; cpt cAMP, 8-(4-chlorophenylthio)-cAMP.
Cytoplasmic Ca2+ concn. (nM) Agonist
Resting
Peak
(10 FZM)
Noradrenaline
(10 4aM) Carbamoylcholine (1 0 ,M) Dibutyryl cAMP (1 mM) cpt cAMP
(1 mM) Ca2+-free medium Isoprenaline
0 0
131.7 + 16.2
170.7 + 18.9***
6.8 + 0.9 (13)
161.6 + 20.3
321.7 + 40.7**
5.4 + 0.8 (8)
162.1 +16.5
278.2 +32.7*
8.0+1.9 (4)
137.5 +15.4
142.3 + 15.2
- (5)
102.0 + 4.7
101.7 + 8.4
- (3)
62.9 + 1 0.5t
94.0 + 12.3**
7.5 + 1.3 (4)
112.0 + 13.3*
5.2 + 0.5 (4)
71.1 + 1 8.6t 109.3 + 1 5.8**
4.7 + 0.7 (4)
68.3 + 2.4t
Carbamoylcholine
extracellular Ca2+ (Figure 1). However, under these conditions, the Ca2' response was transient, returning to resting within approx. 30 s. The responses elicited by noradrenaline and carbamoylcholine were both markedly decreased (to 30-40 nM above resting) in the absence of extracellular Ca2+ (Figures 3 and 4) and, like that for isoprenaline, became transient. Combined data (Table 1) shows that the average resting free Ca2+ concentration was lower (60-70 nM) in the absence of extracellular Ca2+. The data show that all three stimuli have a component of cytoplasmic
--I
I
0
30 Time (s)
60
Figure 4 Noradrenallne Increases cytoplasmic free Ca2+ predominantly by
Ca2+ Influx
Cytoplasmic free Ca2+ concentration was measured in response to 10 uM noradrenaline in KHB ) or Ca2+-free (+0.1 mM EGTA) KHB (----). The traces are representative of at least four experiments.
Table 2 Effect of sequential addtlion of Isoprenallne and carbamoylcholne on cytoplasmic free Ca2+ changes In rat submandibular aclnl Data show changes in free Ca2+ concentration ([Ca2+]) in response to agonists added either as the first stimulus or as second stimulus in the continued presence of the first, after establishment of the plateau phase of the first response (approx. 60 s after addition of the first agonist). Original resting free Ca2+ concentrations were: 155.3 + 38.5 nM (n = 5) in experiments when isoprenaline was added first, and 159.7 + 23.1 nM (n = 3) in experiments when carbamoylcholine was added first. Changes in response to the second agonist were calculated by subtracting the free Ca2+ concentration immediately before addition. Results are means+S.E.M. for the number of experiments shown in parentheses.
Change in
First stimulus
(1 0 FM) Noradrenaline (1 0 FZM)
(10 FM)
>;~ ~ ~ ~ ~ 1
50-
Time to peak (s)
Ca2+-containing medium Isoprenaline
Noradrenaline
Isoprenaline (10 ,uM) Carbamoylcholine (10 ,M)
[Ca2]
(nM)
40.8 + 6.1 (5)
117.2 + 31.9 (3)
Change in
[Ca2+] Second stimulus
(nM)
Carbamoylcholine (10 FM) Isoprenaline (10 FM)
87.1 + 20.9 (5) 50.8 + 9.7 (3)
Ca2+ rise which is independent of extracellular Ca2+. At least 75% of the response to ,-adrenergic stimulation apparently results from mobilization of Ca2+ from an intracellular store(s). This correlates with the finding that approx. 75 % of the mucinsecretion response to isoprenaline was retained on removal of extracellular Ca2+ [6]. Isoprenaline did not increase formation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4 [12], but elevated cyclic AMP levels [1,8]. Table 1 shows that neither dibutyryl cyclic AMP nor 8-(4-chlorophenylthio)-cyclic AMP had an effect on cytoplasmic free Ca2+ concentration, indicating that mobilization of intracellular Ca2+ in response to 8-adrenergic stimulation does not result from an increase in cellular cyclic AMP. In order to investigate whether isoprenaline and carbamoylcholine mobilize Ca2+ from separate stores, changes in free Ca2+ concentration were measured during sequential addition of agonists. As shown in Table 2, the response to either agonist was the same whether it was added first or second. Continued receptor occupancy by the first agonist would be expected to prevent re-accumulation of Ca2+ by the store released by that agonist.
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DISCUSSION The data in the present report demonstrate quantitative changes in cytoplasmic free Ca21 concentration in response to the neurotransmitters which primarily regulate salivary secretion. The study has utilized a preparation of isolated acini which, unlike single cells, show correct polarization of the acinar cell and similar secretory responses to those of undissociated tissue fragments [6]. It has been clearly demonstrated that ,-adrenergic stimulation of submandibular acini, either by isoprenaline or by noradrenaline in the presence of phentolamine, resulted in a rise in cytoplasmic Ca2+ of approx. 40 nM. This is consistent with earlier 45Ca2+ flux data [6,13,14], with a qualitative demonstration using quin2 [15] and with observed changes in cytoplasmic free Ca2+ in response to isoproterenol in other epithelial cell types [16-19]. There is not a simple correlation between the order of effectiveness of agonists in stimulating mucin release and increasing cytoplasmic free Ca21 concentration. The finding that carbamoylcholine and noradrenaline evoked much greater increases in cytoplasmic Ca2+ concentration than did isoprenaline could be related to their actions in markedly stimulating Ca2+mediated fluid secretion [20], which the data suggest requires Ca2+ entry. It is possible to reconcile the lack of quantitative correlation by suggesting that a small component of the total free Ca2+ rise (approx. 40 nM) is sufficient to facilitate the stimulation of secretion. This is consistent with the demonstration [2] that introduction of BAPTA into intact rat submandibular acini inhibited ,-adrenergic stimulation of mucin secretion and supports the hypothesis that Ca2+ is required at a common distal point in the pathways regulating secretion. A generalized rise in cytoplasmic free Ca2+ concentration is, however, not a sufficient trigger [21], since the bivalent-cation ionophore A23187 increased intracellular Ca2+ in submandibular acini without stimulating mucin secretion. The demonstration that cyclic AMP analogues did not evoke a rise in cytoplasmic Ca2+ concentration (Table 1) also suggests that other mechanisms are involved. Cyclic AMP analogues gave the same maximal mucin secretion response as isoprenaline (10 #tM), suggesting that they produce at least as effective an intracellular cyclic AMP concentration [22]. In addition, it can be estimated that the intracellular cyclic AMP concentration after isoprenaline (10 uM) stimulation is approx. 30-40,M [1,2], whereas cyclic AMP analogues were used at 1 mM. Cyclic AMP analogues, acting via protein kinase A, might therefore increase the sensitivity to Ca21 of a Ca2+_ dependent step in the regulatory pathway, allowing stimulation of secretion to occur at resting free Ca2+ concentration. Such a mechanism has been suggested on the basis of interaction between isoprenaline and other secretagogues in parotid and lacrimal acinar cells [23,24]. In dog tracheal cells, there is confficting evidence as to whether cyclic AMP analogues directly increase free Ca2+ concentrations [18,19]. The cellular location ofthe key Ca2+ rise involved in stimulating mucin secretion is not known, but might logically be expected to be at the apical end of the acinar cell. Analysis by single-cell microfluorimetry [25] or confocal scanning microscopy [26] suggests that the initial Ca2+ rise in response to protein secretagogues is at the apical part of the pancreatic acinar cell. In salivary acinar cells, opening of basolateral K+ channels is also required to maintain fluid secretion [20]. In parotid acinar cells, K+-channel opening occurred before a rise in free Ca2+ concentration [16,27], suggesting an initial Ca2+ rise close to the basolateral membrane, followed by a generalized spread of the signal throughout the cell. It will be important to determine whether this pattern of Ca2+ rise is evoked in submandibular
acinar cells by the main fluid-secreting stimuli, noradrenaline (a effect) or carbamoylcholine. The data in the present paper suggest that the large rise in free Ca2+ concentration seen in response to these agonists is mainly dependent on Ca2l entry. Both agonists increase Ins(1,4,5)PJ formation in submandibular acini [12,28], suggesting the possibility of a link between Ca2l release from an Ins(1,4,5)P3-sensitive store and Ca2l entry. The data showing that, after carbamoylcholine stimulation, the ,agonist isoprenaline elicited a normal Ca2l rise (Table 2) strongly suggest that isoprenaline and carbamoylcholine act on functionally distinct intracellular Ca2+ stores. The data do not, however, allow conclusions as to whether the stores are located in different organelles. Intracellular Ca2+ mobilization resulting from ,3-adrenergic stimulation is therefore likely to be from a localized Ins(1,4,5)P3insensitive store. It is conceivable that ,-adrenergic stimulation causes a rise in Ins(1,4,5)P3 concentration too small or too localized to be detected by the methods used [12], yet sufficient to cause Ca2+ release. It has been demonstrated in permeabilized hepatocytes [29] that protein kinase A can sensitize the Ins(1,4,5)P3 receptor; such a mechanism could explain Ca2+ mobilization in response to ,-adrenergic stimulation without a change in Ins(1,4,5)P3 concentration. However, noradrenaline and isoprenaline (each at 10 #M) elicited the same degree of Ca2+ mobilization (Table 1), yet isoprenaline (10 saM) gave no increase in Ins(1,4,5)P3 formation, whereas a 4-fold increase was seen with noradrenaline (10 ,uM) [12]. Furthermore, the Ca2+ signal elicited by noradrenaline in the presence of phentolamine was inhibited by propranolol (Figure 2), whereas stimulation of Ins(1,4,5)P3 formation by noradrenaline was not affected by propranolol [12]. Thus it is probable that another messenger is required for ,adrenergic stimulation of Ca2+ mobilization. Potential candidates include cyclic GMP and cyclic ADP-ribose, the latter having been shown to increase Ca2+ release in other cell types [30]. In summary, it has been shown that /3-adrenergic stimulation of submandibular acini results in a rise in cytoplasmic free Ca2+ concentration which is in part derived from an inositol phosphateinsensitive store. Although the degree of stimulation of mucin secretion does not quantitatively correlate with the maximum rise in Ca2+ concentration, the data are consistent with the hypothesis that a small rise in Ca2+ concentration plays a key role in stimulating exocytosis. We thank the Cystic Fibrosis Trust (U.K.) and the Arthritis and Rheumatism Council for financial support.
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Received 19 November 1992/8 March 1993; accepted 16 March 1993
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