0003-9969/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
Archs oral Biol. Vol. 36, No. 5, pp. 335-340, 1991 Printed in Great Britain. All rights reserved
RESPONSE
OF RAT PAROTID
ACINI TO BARIUM
J. P. DEHAYE, C. CAULIER and C. DELP~RTE Department of Biochemistry, Faculty of Medicine, Free University of Brussels, 2, rue Evers, BlOOOBrussels, Belgium (Accepted 9 January 1991)
Summary-The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of “‘Ca2+but , at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholin’e-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion.
Key words: Calcium-activated
potassium channel, exocytosis, amylase, carbamylcholine,
IN’I’RODUCTION
Parasympathetic stimulation of parotid glands elicits the secretion of a copious flow of dilute saliva (Putney, 1986a). This secretion is initiated by the activation of a polyphosphatidylinositol-specific phospholipase C, subsequent to the occupancy of receptors by their agonists (Aub and Putney, 1985). The hydrolysis of the phospholipid generates two signals, inositol 1,4,5_trisphosphate responsible for the mobilization of intracellular calcium (Takemura, 1985), and diglycerides, which activate the phospholipid-sensitive, calcium-dependent, protein kinase C (Dowd et nl., 1987). The increase in the cytosolic calcium concentration activates the exocytosis and increases the probability that calcium-dependent potassium channels located at the baso-lateral side of the acinar cell will be open (Maruyama, Gallacher and Petersen, 1983). Similar channels have been described in other secretory tissues like the pig exocrine pancreas (Iwatsuki and Petersen, 1985), the rat lacrimal gland (Trautmann and Marty, 1984). and anteriorhypophysis (Ritchie, 1987). In the parotid gland, the opening of these calciumactivated potassium channels is coupled with the opening of apical chloride channels, and the acinar cells lose potassium chloride and shrink (Foskett and Melvin, 1989). After the massive extrusion of potassium (Duner-Engstriim, Larsson and Fredholm, 1986), this ion is secondarily taken back by two mechanisms, a sodium,-potassium chloride co-transport system (Helman et al., 1987) and a sodiumpotassium pump (Hootman and Williams, 1985). The result of these events is a net uptake of chloride, Abbreviation:
EGTA: ether)N,N’-tetraacetic
ethyleneglycol-bis(/?-amino-ethyl acid.
substance P.
which flows through the cell towards the apical pole where it is extruded. The outflow of chloride is finally responsible for the efflux of sodium and water from the gland (Melvin et al., 1987). The potassium channels are thus primarily involved in saliva formation. In spite of this crucial role, little is known about the biochemistry and the pharmacology of these channels. Electrophysiological studies have revealed that they have a high conductance (250 pS) and a high selectivity with respect to potassium. They are activated by calcium concentrations in the 0.1-l PM range, and depolarization increases the sensitivity of these channels towards calcium. Each acinar cell has only a small number of these channels (less than 100 per cell-Iwatsuki et al., 1985; Maruyama et al., 1986). We have recently reported that they are insensitive to apamin, a well-known potassium channel blocker, and that they are blocked by the venom from Leiurus quinquestriatus (Dehaye, Winand and Christophe, 1987), presumably by the charybdotoxin present in this venom (Smith, Phillips and Miller, 1986). Barium is a divalent cation that can block the calcium-dependent potassium channels in various tissues such as the squid giant synapse (Augustine, Charlton and Horn, 1988), the medullary thick ascending limb cells (Guggino et al., 1987), intestinal smooth-muscle cells (Benham ef al., 1985) or the exocrine pancreas (Iwatsuki and Petersen, 1985). Our purpose now was to test the effect of barium on “Rb+ efflux in rat parotid acini. It has also been reported that barium could substitute for calcium in secretory processes (Grill and Efendic, 1984) contractions of and in calmodulin-dependent vascular muscles (Kreye, Hofmann and Miilheisen, 1986) and so we tested the effect of this ion on exocytosis. 335
J. P. DEHAYE et al.
336 MATERIALS
AND METHODS
Male Wistar rats (1 SO-200 g), fed ad Zibitum, were used. Substance P and carbamylcholine were from Sigma Chemical Company (St Louis, MO); 45Ca2+ was supplied by Amersham International plc (Amersham, Bucks., U.K.) and 86Rb+ by New England Nuclear (Dreieich, Germany); collagenase CLSPA was purchased from Worthington (Freehold, NJ). After decapitation of the animals, their parotid glands were excised and the acini were prepared, as described by Dehaye et al. (1985). The isolated acini were resuspended in fresh incubation medium and 200 ~1 fractions of this suspension were incubated for 20min at 37°C in the presence of the tested agents. At the end of the incubation, the acini were centrifuged for 10 s in a Beckman 152 microfuge. The amylase present in the supernatant was assayed according to Noelting and Bernfeld (1948). Non-incubated samples were used to estimate the amylase already present in the medium at the beginning of the incubation. Fractions of the acini suspension were diluted with fresh medium, sonicated and assayed in order to estimate the total amylase content of the suspension. Results were expressed as percentage of total amylase content released in the medium during the incubation. Acini isolated from four glands were preincubated in 2.5 ml incubation medium at 37°C in the presence of 20 pCi/ml 45Ca2+ or 86Rb+. The suspension was washed twice with an isotope-free medium and resuspended in 7ml fresh medium. Fractions (200~1) of this suspension were incubated for 2min at 37°C in the presence of the tested agents. The acini were then centrifuged for 5 s at 10,OOOg in a Beckman 152 microfuge and 75 ~1 of the supernatant were counted in a Beckman spectrometer. Results were corrected for the radioactivity already present in the medium at the beginning of the experiment and were expressed as percentage of residual intracellular isotope released during the 2-min experiment.
OL
-8
-7 -6 [Carbamylcholinel
-5
-4
(Log H)
Fig. 1. Effect of barium ion on the doseeresponse curve of carbamylcholine on *6Rb+ efflux. The acini were preloaded with the isotope, washed and incubated for 2min in the presence of various concentrations of carbamylcholine and in the absence ( x ) or in the presence of barium ion at a 1 mM (O), 3 mM (0) or 10 mM (A). The results were expressed as percent residual intracellular isotope released within 2 min. They are the means IfI SEM of four exper-
iments. concentrations of barium higher than 100 PM inhibited carbamylcholine-stimulated @Rb+ efflux. At a maximal 10mM barium concentration, this inhibition was around 50%, independent of the concentration of carbamylcholine used [Fig. 2(A)]. Substance P at a 10 nM concentration doubled *‘Rb+ efflux, increased this efflux 3.5-fold at a 0.1 PM concentration and 4-fold at a 1 PM concentration [Fig. 2(B)]. Barium concentrations higher than 100pM inhibited the efflux of 86Rb+ stimulated by the undecapeptide [Fig. 2(B)] and at a 10 mM concentration, barium inhibited by 50% the response to 1 PM substance P. In the previous experiments, the effect of barium was tested in the presence of 0.5 mM extracellular calcium. The inhibitory effect of barium was tested next in the absence of extracellular calcium. The acini were resuspended in a medium containing a small (0.1 mM) calcium concentration, which prevented a severe depletion of intracellular calcium before starting the experiment. The acini were then incubated in
RESULTS
As shown in Fig. 1, the loaded acini lost about 15% of their total 86Rbf content within 2min of incubation in control conditions. Carbamylcholine increased the efflux of 86Rb+ when tested in the 0.1-100 PM concentration range. Half-maximal concentration was 1 PM and the maximal effect (a 3.5-fold increase) was observed at 100 PM. Barium at a 1 mM concentration slightly inhibited (- 20%) the effect of carbamylcholine, but, at a 3 mM concentration, barium inhibited by 40% the response to carbamylcholine without affecting the half-maximal concentration of the muscarinic agonist. This suggested that the inhibition was purely non-competitive. Further inhibition (60%) was observed when barium was used at a 10mM concentration. In the next experiment, the inhibitory effect of barium on the 86Rb+ efflux induced by three concentrations of carbamylcholine [Fig. 2(A)] was compared with its effect on the response to substance P [Fig. 2(B)]. Barium concentrations in the 0.1-10 mM range did not affect basal “Rb+ efflux but
oh+------5
-4
-3
-2 iBarIum
041
-5
-4
-3
-2
(Log Mi
Fig. 2. Effect of increasing concentrations of barium ion on XhRb+ efflux stimulated by carbamylcholine (A) or substance P (B). After the preloading period and washing, the acini were incubated for 2min in the presence of increasing concentrations of barium. in control conditions (x) or in the presence of (A) carbamylcholine 0.1 p M (O), 1 p M (A) or 10 PM (0) or (B) substance P 10 nM (0). 0.1 PM (A) or 1 PM (0). The results were expressed as percentage residual intracellular isotope released in the medium within 2 min. They are the means + SEM of five experiments.
Barium and rat parotid gland
the same medium, but in the presence of 0.5 mM EGTA and in the presence of various concentrations of added calcium. As shown in Fig. 3, incubating the acini in the presence of 0.1 mM calcium and 0.5 mM EGTA (i.e. in the virtual absence of free extracellular calcium) did not prevent the stimulator-y effect of carbamylcholine on %Rb+ efflux. Adding increasing concentrations of extracellular calcium further increased the efflux of “Rb+, both in basal conditions and in the presence of low (0.1 and 1 p M) carbamylcholine concentrations. At a 10 mM calcium concentration, the efflux of s6Rb+ was increased by 25% in basal conditions and only by 5% in the presence of 10pM carbamylcholine. A similar experiment was performed with barium. The acini were incubated in the presence of 0.1 mM extracellular calcium and 0.5 mM EGTA, and various concentrations of barium (from 10 PM to 10mM) were added. In these conditions, barium did not affect the basal release of *6Rb+ but at concentrations higher than 100 PM still inhibited the efflux of the isotope stimulated by carbamylcholine. The fact that barium could inhibit carbamylcholine- as well as substance P-stimulated *‘Rb+ efflux suggested that the divalent cation acted distally to the plasma membrane receptors. In order to confirm that it blocked the calcium-activated potassium channels and not some intracellular response subsequent to receptor occupancy, the effect of barium on intracellular calcium mobilization was tested. The acini were loaded with 45Ca2+, washed and incubated for 2 min at 37°C. In these conditions, the acini released 4% of their intracellular 4sCa2f in basal conditions (Fig. 4). Carbamylcholine at concentrations higher than 0.1 PM stimulated the efflux of 45Ca2+. Half-maximal effect was observed at a 3 PM concentration and the maximal response (an 8-fold increase) at a 100 PM concentration (Fig. 4). Barium at a 10 mM concentration did not affect 45Ca2+efflux in basal conditions nor in the presence of various concentrations of carbamylcholine.
oL-------
-4 -3 -2 Kalciunl (Log Ml
331
[Carbalylcholinel
(Log Ml
Fig. 4. Effect of barium on the dose-response curves of carbamylcholine on Ca2+ efflux and amylase release. (A) The acini were preloaded with ‘%a*+, washed and incubated in the presence of various concentrations of carbamylcholine, in the absence (0) or in the presence (a) of 10 mM barium. Results were expressed as percent residual intracellular isotope released within 2 min. They are the means * SEM of four experiments. (B) The acini were incubated for 20min in the presence of various concentrations of carbamylcholine and in the absence (A) or in the presence (A) of 10 mM barium. Results were expressed as total amylase released in the medium within 20 min. They are the means + SEM of five experiments.
The efflux of 45Ca2+ was next measured in the presence of various concentrations of extracellular calcium or barium. Removal of free extracellular calcium did not affect basal or carbamylcholinestimulated 45Ca2+ efflux. Increasing the concentrations of extracellular calcium or barium had no effect on the etIIux of the isotope (Fig. 5). The effect of barium on amylase secretion was tested next. Rat parotid acini released 1.5% of their total amylase content within 20min. Increasing the concentration of carbamylcholine-stimulated exocytosis. Half-maximal stimulation occurred at a 0.3 PM concentration. The maximal effect (a 5-fold increase) was observed at a 10 p M carbamylcholine concentration (Fig. 4). Barium at a 10mM concentration increased basal amylase release by 70%. It did not affect the secretory response to carbamylcholine. The amylase secretion was very sensitive to variations in the extracellular concentration of calcium. In the presence of 0.1 mM calcium and 0.5 mM EGTA, carbamylcholine could still stimulate amylase
OL+-----
-4 -3 -2 IBarium (Log 111
Fig. 3. ERect of increasing concentrations of calcium (A) and barium (B) on s6Rb+ efflux stimulated by carbamvlcholine. After the ireloading period and the washing, the acini were resuspended in a medium containing 0.1 mM calcium, and incubated for 2 min in the presence of 0.5 mM EGTA and of various concentrations of calcium (A) or barium (B). These incubations were performed in control conditions ( x ), or in the presence of carbamylcholine 0.1 PM (O), t PM (A) or IOhM (0). Results were expressed as percent residual intracellular isotope released within 2 min. They are the means + SEM of five experiments.
10
10
+ t---l-*-r-f’ -
0t
I
_30+-----4 [Calciunl (Log Ml
--I k--r.,_r-&--r t -4 -3 -2 IBarium ILog Ml
Fig. 5. Eti’ect of various concentrations of calcium (A) and barium (B) on Ca2+ efflux stimulated by carbamylcholine. The acini were incubated as described in Fig. 3. Results were expressed as percent residual intracellular isotope released in the medium within 2 min. They are the means f SEM of five experiments.
J. P. DEHAYE et al.
338
ICalciual
ILog I()
Ktariual
(Log r0
Fig. 6. Effect of increasing concentrations of calcium (A) and barium (B) on amylase secretion stimulated by carbamylcholine. The acini were incubated for 20 min as described in Fig. 3. Results were expressed as percent total amylase released in the medium within 20 min. They are the means f SEM of five experiments.
(a 7-fold stimulation at 10 PM, Fig. 6). This secretion was probably due to the mobilization of intracellular stores of calcium and to the activation of protein kinase C. But when extracellular calcium concentrations were higher than the concentration of EGTA in the extracellular medium, the response to carbamylcholine was potentiated (from 6.9% at 0.1 mM calcium to 12.6% in the presence of 1.l mM calcium, at a 10 FM carbamylcholine concentration). Increasing the extracellular calcium concentration above 1 mM not only further increased carbamylcholine-stimulated amylase release but also increased basal amylase release (from 1.3% at 0.1 mM calcium to 5.7% at 10 mM calcium). Barium had an effect similar to that of calcium: high concentrations of barium (above 1 mM) increased basal amylase release while, in the presence of carbamylcholine, lower concentrations of barium (in the 0.1-l mM range) were stimulatory. At all concentrations tested, barium was less efficient than calcium, e.g. in basal conditions and in the presence of 10mM calcium, 5.7% of total amylase were released while only 2.4% were released in the presence of the same concentration of barium. secretion
DISCUSSION
We report that concentrations of barium in the millimolar range can inhibit the release of 86Rb+ in response to carbamylcholine or substance P. This inhibition was observed in the presence or in the absence of extracellular calcium, suggesting that barium did not compete with extracellular calcium. As barium inhibited carbamylcholine- as well as substance P-induced isotope efflux, its action must be distal to plasma membrane receptors. This was confirmed by its lack of effect on 45Ca2+efflux, which also implied that barium did not interfere with the mobilization of intracellular pools of calcium. These findings suggest that barium directly inhibited the calcium-activated potassium channels. This inhibition was non-competitive and, at a 10 mM barium concentration, about 50% of the channels were blocked. This is in agreement with the electrophysiological studies of Iwatsuki and Petersen (1985), who showed that barium could block calcium-activated
potassium channels in pig pancreas, and are consistent with the present view that barium is a general inhibitor of such channels when used in the millimolar range. It remains to be established on which site of the channel barium acts. It has recently been reported that barium inhibits the release of amylase induced by CCK-8 from permeabilized rat pancreatic acini but not from intact acini (Fuller, Eckhardt and Schulz, 1989). This has been interpreted as an evidence for the presence of calcium-activated potassium channels on the membrane of zymogen granules. The blockade of these channels by barium prevents the entry of potassium (and its counter-anion, chloride) in the granules and is likely to prevent the osmotic swelling of these granules. This finding also suggests that barium cannot cross an intact plasma membrane. On the other hand, barium can enter the chromaffin cells through a channel for divalent cations (Artalejo, Garcia and Aunis, 1987). More recently, it has been reported that barium could enter inside lacrimal glands via the calcium channel activated by agonists (Kwan and Putney, 1990). Similar channels have been described in the parotid gland (Takemura and Putney, 1989), where they are activated by carbamylcholine (Mertz er al., 1990). This could explain why barium is a better inhibitor of calcium-activated potassium channels (see Fig. 2) and of salivary secretion (Young et al., 1987) in the presence of high concentrations of carbamylcholine. It has also been demonstrated that barium could block *6Rb+ influx in inside-out vesicles from kidney cells (Klaerke, Karlish and Jorgensen, 1987). It is thus possible that it blocks the potassium channel after binding to a site located on the cytoplasmic side of the channel. One possibility would be that barium binds to and blocks the calcium-binding site of the channel. Such a hypothesis is consistent with the fact that barium blocks some intracellular calcium-activated enzymes (Warchek et al., 1987). For secretion, the action of barium is opposite to that described above as it can mimic the action of extracellular calcium and stimulate amylase secretion. This is even more obvious in our experiments where extracellular calcium had been chelated with EGTA. Some important conclusions can be drawn from such experiments. First, removal of extracellular calcium did not affect the efflux of 86Rb+, confirming that the efflux of the isotope is strictly dependent (at least in short experiments) on the mobilization of an intracellular pool of calcium (Foskett et al., 1989). Second, the efflux of 45Ca2+ was not modified by the absence of calcium in the extracellular medium, showing that a Ca/Ca exchange is not involved in the extrusion of 45Ca2+. Third, the basal amylase release could be stimulated by the increase in the extracellular calcium concentration. In rat parotid acini, calcium is thus a potent secretagogue by itself and does not require the activation of protein kinase C by diglycerides to stimulate amylase release. Increasing the extracellular calcium concentration also potentiated the secretory response to carbamylcholine. Two explanations can account for this observation: (1) amylase secretion was measured after 20 min, and the acini have thus been exposed to EGTA for this long period, which is likely to deplete intracellular stores of calcium and to decrease the response to the muscarinic agent; (2) it
Barium and rat parotid gland should also be recalled that within 20min, the response to carbamylcholine relies not only on intracellular stores of calcium, but also on extracellular calcium, the influx of which is stimulated by the depletion of the IP,-sensitive intracellular store (Putney, 1986b). It remains that increasing the extracellular concentration of calcium above 1 mM activates amylase secretion. In the absence of free extracellular calcium, barium inhibited the efflux of *‘Rb+ but did not affect the efflux of 4SCa2+.It could still stimulate amylase release, albeit at a lower rate than calcium. Our ‘calcium-free’ medium contained 0.1 mM calcium and 0.5 mM EGTA, and the apparent stimulatory effect of barium might be secondary to the displacement of the calcium bound to EGTA. However, this is unlikely because the effect of high concentrations of barium is greater than the effect observed in the presence of EGTA 0.5 mM and 1.1 mM calcium, which would lead to a free calcium concentration higher than 0.1 mM. In conclusion, barium has two effects in rat parotid acini. It is an antagonist of the calcium-activated potassium channels and an agonist on amylase secretion. Acknowledgement-This
work was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique.
REFERENCES
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Benham C. D., Bolton T. B., Lang R. J. and Takewaki T. (1985) The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes. Pftigers Arch. 403, 120-127.
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Dehaye J. P., Winand J. and Christophe J. (1987) Scorpion venom inhibits carbamylcholine-induced %ubidium efflux from rat parotid acini. FEBS L&t. 219, 451454. Dowd F., Watson E. L., Lau Y.-S., Justin J., Pasieunik J. and Jacobson K. L. (1987) Calcium-dependent protein kinase reactions associated with parotid gland secretory granule membranes. J. dent. Res: 66, 557-563. _ Duner-Enestriim M.. Larsson 0. and Fredholm B. B. (1986) Continu&s monitoring of potassium efflux from rat parotid gland fragments by potassium selective electrodes. Acta physiol. stand. 126, 551-555. Foskett J. K. and Melvin J. E. (1989) Activation of salivary secretion: coupling of cell volume and [Ca2+], in single cells. Science 244, 1582-1585. Foskett J. K., Gunther-Smith P. J., Melvin J. E. and Turner R. J. (1989)Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells. Proc. natn. Acad. Sci. 86, 167-171.
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Fuller C. M., Eckhardt L. and Schulz I. (1989) Ionic and osmotic dependence of secretion from permeabilised acini of the rat pancreas. PJtigers Arch. 413, 385-394. Grill V. and Efendic S. (1984) Stimulation by calcium and
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Smith C., Phillips M. and Miller C. (1986) Purification of charybdotoxin, a specific inhibitor of the high-conductance Ca2+-activated K+ channel. J. biol. Chem. 261, 14607-14613. Takemura H. (1985) Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholin-
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Archs oral Biol. Vol. 36, No. 5, pp. 335-340, 1991 Printed in Great Britain. All rights reserved
RESPONSE
OF RAT PAROTID
ACINI TO BARIUM
J. P. DEHAYE, C. CAULIER and C. DELP~RTE Department of Biochemistry, Faculty of Medicine, Free University of Brussels, 2, rue Evers, BlOOOBrussels, Belgium (Accepted 9 January 1991)
Summary-The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of “‘Ca2+but , at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholin’e-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion.
Key words: Calcium-activated
potassium channel, exocytosis, amylase, carbamylcholine,
IN’I’RODUCTION
Parasympathetic stimulation of parotid glands elicits the secretion of a copious flow of dilute saliva (Putney, 1986a). This secretion is initiated by the activation of a polyphosphatidylinositol-specific phospholipase C, subsequent to the occupancy of receptors by their agonists (Aub and Putney, 1985). The hydrolysis of the phospholipid generates two signals, inositol 1,4,5_trisphosphate responsible for the mobilization of intracellular calcium (Takemura, 1985), and diglycerides, which activate the phospholipid-sensitive, calcium-dependent, protein kinase C (Dowd et nl., 1987). The increase in the cytosolic calcium concentration activates the exocytosis and increases the probability that calcium-dependent potassium channels located at the baso-lateral side of the acinar cell will be open (Maruyama, Gallacher and Petersen, 1983). Similar channels have been described in other secretory tissues like the pig exocrine pancreas (Iwatsuki and Petersen, 1985), the rat lacrimal gland (Trautmann and Marty, 1984). and anteriorhypophysis (Ritchie, 1987). In the parotid gland, the opening of these calciumactivated potassium channels is coupled with the opening of apical chloride channels, and the acinar cells lose potassium chloride and shrink (Foskett and Melvin, 1989). After the massive extrusion of potassium (Duner-Engstriim, Larsson and Fredholm, 1986), this ion is secondarily taken back by two mechanisms, a sodium,-potassium chloride co-transport system (Helman et al., 1987) and a sodiumpotassium pump (Hootman and Williams, 1985). The result of these events is a net uptake of chloride, Abbreviation:
EGTA: ether)N,N’-tetraacetic
ethyleneglycol-bis(/?-amino-ethyl acid.
substance P.
which flows through the cell towards the apical pole where it is extruded. The outflow of chloride is finally responsible for the efflux of sodium and water from the gland (Melvin et al., 1987). The potassium channels are thus primarily involved in saliva formation. In spite of this crucial role, little is known about the biochemistry and the pharmacology of these channels. Electrophysiological studies have revealed that they have a high conductance (250 pS) and a high selectivity with respect to potassium. They are activated by calcium concentrations in the 0.1-l PM range, and depolarization increases the sensitivity of these channels towards calcium. Each acinar cell has only a small number of these channels (less than 100 per cell-Iwatsuki et al., 1985; Maruyama et al., 1986). We have recently reported that they are insensitive to apamin, a well-known potassium channel blocker, and that they are blocked by the venom from Leiurus quinquestriatus (Dehaye, Winand and Christophe, 1987), presumably by the charybdotoxin present in this venom (Smith, Phillips and Miller, 1986). Barium is a divalent cation that can block the calcium-dependent potassium channels in various tissues such as the squid giant synapse (Augustine, Charlton and Horn, 1988), the medullary thick ascending limb cells (Guggino et al., 1987), intestinal smooth-muscle cells (Benham ef al., 1985) or the exocrine pancreas (Iwatsuki and Petersen, 1985). Our purpose now was to test the effect of barium on “Rb+ efflux in rat parotid acini. It has also been reported that barium could substitute for calcium in secretory processes (Grill and Efendic, 1984) contractions of and in calmodulin-dependent vascular muscles (Kreye, Hofmann and Miilheisen, 1986) and so we tested the effect of this ion on exocytosis. 335
J. P. DEHAYE et al.
336 MATERIALS
AND METHODS
Male Wistar rats (1 SO-200 g), fed ad Zibitum, were used. Substance P and carbamylcholine were from Sigma Chemical Company (St Louis, MO); 45Ca2+ was supplied by Amersham International plc (Amersham, Bucks., U.K.) and 86Rb+ by New England Nuclear (Dreieich, Germany); collagenase CLSPA was purchased from Worthington (Freehold, NJ). After decapitation of the animals, their parotid glands were excised and the acini were prepared, as described by Dehaye et al. (1985). The isolated acini were resuspended in fresh incubation medium and 200 ~1 fractions of this suspension were incubated for 20min at 37°C in the presence of the tested agents. At the end of the incubation, the acini were centrifuged for 10 s in a Beckman 152 microfuge. The amylase present in the supernatant was assayed according to Noelting and Bernfeld (1948). Non-incubated samples were used to estimate the amylase already present in the medium at the beginning of the incubation. Fractions of the acini suspension were diluted with fresh medium, sonicated and assayed in order to estimate the total amylase content of the suspension. Results were expressed as percentage of total amylase content released in the medium during the incubation. Acini isolated from four glands were preincubated in 2.5 ml incubation medium at 37°C in the presence of 20 pCi/ml 45Ca2+ or 86Rb+. The suspension was washed twice with an isotope-free medium and resuspended in 7ml fresh medium. Fractions (200~1) of this suspension were incubated for 2min at 37°C in the presence of the tested agents. The acini were then centrifuged for 5 s at 10,OOOg in a Beckman 152 microfuge and 75 ~1 of the supernatant were counted in a Beckman spectrometer. Results were corrected for the radioactivity already present in the medium at the beginning of the experiment and were expressed as percentage of residual intracellular isotope released during the 2-min experiment.
OL
-8
-7 -6 [Carbamylcholinel
-5
-4
(Log H)
Fig. 1. Effect of barium ion on the doseeresponse curve of carbamylcholine on *6Rb+ efflux. The acini were preloaded with the isotope, washed and incubated for 2min in the presence of various concentrations of carbamylcholine and in the absence ( x ) or in the presence of barium ion at a 1 mM (O), 3 mM (0) or 10 mM (A). The results were expressed as percent residual intracellular isotope released within 2 min. They are the means IfI SEM of four exper-
iments. concentrations of barium higher than 100 PM inhibited carbamylcholine-stimulated @Rb+ efflux. At a maximal 10mM barium concentration, this inhibition was around 50%, independent of the concentration of carbamylcholine used [Fig. 2(A)]. Substance P at a 10 nM concentration doubled *‘Rb+ efflux, increased this efflux 3.5-fold at a 0.1 PM concentration and 4-fold at a 1 PM concentration [Fig. 2(B)]. Barium concentrations higher than 100pM inhibited the efflux of 86Rb+ stimulated by the undecapeptide [Fig. 2(B)] and at a 10 mM concentration, barium inhibited by 50% the response to 1 PM substance P. In the previous experiments, the effect of barium was tested in the presence of 0.5 mM extracellular calcium. The inhibitory effect of barium was tested next in the absence of extracellular calcium. The acini were resuspended in a medium containing a small (0.1 mM) calcium concentration, which prevented a severe depletion of intracellular calcium before starting the experiment. The acini were then incubated in
RESULTS
As shown in Fig. 1, the loaded acini lost about 15% of their total 86Rbf content within 2min of incubation in control conditions. Carbamylcholine increased the efflux of 86Rb+ when tested in the 0.1-100 PM concentration range. Half-maximal concentration was 1 PM and the maximal effect (a 3.5-fold increase) was observed at 100 PM. Barium at a 1 mM concentration slightly inhibited (- 20%) the effect of carbamylcholine, but, at a 3 mM concentration, barium inhibited by 40% the response to carbamylcholine without affecting the half-maximal concentration of the muscarinic agonist. This suggested that the inhibition was purely non-competitive. Further inhibition (60%) was observed when barium was used at a 10mM concentration. In the next experiment, the inhibitory effect of barium on the 86Rb+ efflux induced by three concentrations of carbamylcholine [Fig. 2(A)] was compared with its effect on the response to substance P [Fig. 2(B)]. Barium concentrations in the 0.1-10 mM range did not affect basal “Rb+ efflux but
oh+------5
-4
-3
-2 iBarIum
041
-5
-4
-3
-2
(Log Mi
Fig. 2. Effect of increasing concentrations of barium ion on XhRb+ efflux stimulated by carbamylcholine (A) or substance P (B). After the preloading period and washing, the acini were incubated for 2min in the presence of increasing concentrations of barium. in control conditions (x) or in the presence of (A) carbamylcholine 0.1 p M (O), 1 p M (A) or 10 PM (0) or (B) substance P 10 nM (0). 0.1 PM (A) or 1 PM (0). The results were expressed as percentage residual intracellular isotope released in the medium within 2 min. They are the means + SEM of five experiments.
Barium and rat parotid gland
the same medium, but in the presence of 0.5 mM EGTA and in the presence of various concentrations of added calcium. As shown in Fig. 3, incubating the acini in the presence of 0.1 mM calcium and 0.5 mM EGTA (i.e. in the virtual absence of free extracellular calcium) did not prevent the stimulator-y effect of carbamylcholine on %Rb+ efflux. Adding increasing concentrations of extracellular calcium further increased the efflux of “Rb+, both in basal conditions and in the presence of low (0.1 and 1 p M) carbamylcholine concentrations. At a 10 mM calcium concentration, the efflux of s6Rb+ was increased by 25% in basal conditions and only by 5% in the presence of 10pM carbamylcholine. A similar experiment was performed with barium. The acini were incubated in the presence of 0.1 mM extracellular calcium and 0.5 mM EGTA, and various concentrations of barium (from 10 PM to 10mM) were added. In these conditions, barium did not affect the basal release of *6Rb+ but at concentrations higher than 100 PM still inhibited the efflux of the isotope stimulated by carbamylcholine. The fact that barium could inhibit carbamylcholine- as well as substance P-stimulated *‘Rb+ efflux suggested that the divalent cation acted distally to the plasma membrane receptors. In order to confirm that it blocked the calcium-activated potassium channels and not some intracellular response subsequent to receptor occupancy, the effect of barium on intracellular calcium mobilization was tested. The acini were loaded with 45Ca2+, washed and incubated for 2 min at 37°C. In these conditions, the acini released 4% of their intracellular 4sCa2f in basal conditions (Fig. 4). Carbamylcholine at concentrations higher than 0.1 PM stimulated the efflux of 45Ca2+. Half-maximal effect was observed at a 3 PM concentration and the maximal response (an 8-fold increase) at a 100 PM concentration (Fig. 4). Barium at a 10 mM concentration did not affect 45Ca2+efflux in basal conditions nor in the presence of various concentrations of carbamylcholine.
oL-------
-4 -3 -2 Kalciunl (Log Ml
331
[Carbalylcholinel
(Log Ml
Fig. 4. Effect of barium on the dose-response curves of carbamylcholine on Ca2+ efflux and amylase release. (A) The acini were preloaded with ‘%a*+, washed and incubated in the presence of various concentrations of carbamylcholine, in the absence (0) or in the presence (a) of 10 mM barium. Results were expressed as percent residual intracellular isotope released within 2 min. They are the means * SEM of four experiments. (B) The acini were incubated for 20min in the presence of various concentrations of carbamylcholine and in the absence (A) or in the presence (A) of 10 mM barium. Results were expressed as total amylase released in the medium within 20 min. They are the means + SEM of five experiments.
The efflux of 45Ca2+ was next measured in the presence of various concentrations of extracellular calcium or barium. Removal of free extracellular calcium did not affect basal or carbamylcholinestimulated 45Ca2+ efflux. Increasing the concentrations of extracellular calcium or barium had no effect on the etIIux of the isotope (Fig. 5). The effect of barium on amylase secretion was tested next. Rat parotid acini released 1.5% of their total amylase content within 20min. Increasing the concentration of carbamylcholine-stimulated exocytosis. Half-maximal stimulation occurred at a 0.3 PM concentration. The maximal effect (a 5-fold increase) was observed at a 10 p M carbamylcholine concentration (Fig. 4). Barium at a 10mM concentration increased basal amylase release by 70%. It did not affect the secretory response to carbamylcholine. The amylase secretion was very sensitive to variations in the extracellular concentration of calcium. In the presence of 0.1 mM calcium and 0.5 mM EGTA, carbamylcholine could still stimulate amylase
OL+-----
-4 -3 -2 IBarium (Log 111
Fig. 3. ERect of increasing concentrations of calcium (A) and barium (B) on s6Rb+ efflux stimulated by carbamvlcholine. After the ireloading period and the washing, the acini were resuspended in a medium containing 0.1 mM calcium, and incubated for 2 min in the presence of 0.5 mM EGTA and of various concentrations of calcium (A) or barium (B). These incubations were performed in control conditions ( x ), or in the presence of carbamylcholine 0.1 PM (O), t PM (A) or IOhM (0). Results were expressed as percent residual intracellular isotope released within 2 min. They are the means + SEM of five experiments.
10
10
+ t---l-*-r-f’ -
0t
I
_30+-----4 [Calciunl (Log Ml
--I k--r.,_r-&--r t -4 -3 -2 IBarium ILog Ml
Fig. 5. Eti’ect of various concentrations of calcium (A) and barium (B) on Ca2+ efflux stimulated by carbamylcholine. The acini were incubated as described in Fig. 3. Results were expressed as percent residual intracellular isotope released in the medium within 2 min. They are the means f SEM of five experiments.
J. P. DEHAYE et al.
338
ICalciual
ILog I()
Ktariual
(Log r0
Fig. 6. Effect of increasing concentrations of calcium (A) and barium (B) on amylase secretion stimulated by carbamylcholine. The acini were incubated for 20 min as described in Fig. 3. Results were expressed as percent total amylase released in the medium within 20 min. They are the means f SEM of five experiments.
(a 7-fold stimulation at 10 PM, Fig. 6). This secretion was probably due to the mobilization of intracellular stores of calcium and to the activation of protein kinase C. But when extracellular calcium concentrations were higher than the concentration of EGTA in the extracellular medium, the response to carbamylcholine was potentiated (from 6.9% at 0.1 mM calcium to 12.6% in the presence of 1.l mM calcium, at a 10 FM carbamylcholine concentration). Increasing the extracellular calcium concentration above 1 mM not only further increased carbamylcholine-stimulated amylase release but also increased basal amylase release (from 1.3% at 0.1 mM calcium to 5.7% at 10 mM calcium). Barium had an effect similar to that of calcium: high concentrations of barium (above 1 mM) increased basal amylase release while, in the presence of carbamylcholine, lower concentrations of barium (in the 0.1-l mM range) were stimulatory. At all concentrations tested, barium was less efficient than calcium, e.g. in basal conditions and in the presence of 10mM calcium, 5.7% of total amylase were released while only 2.4% were released in the presence of the same concentration of barium. secretion
DISCUSSION
We report that concentrations of barium in the millimolar range can inhibit the release of 86Rb+ in response to carbamylcholine or substance P. This inhibition was observed in the presence or in the absence of extracellular calcium, suggesting that barium did not compete with extracellular calcium. As barium inhibited carbamylcholine- as well as substance P-induced isotope efflux, its action must be distal to plasma membrane receptors. This was confirmed by its lack of effect on 45Ca2+efflux, which also implied that barium did not interfere with the mobilization of intracellular pools of calcium. These findings suggest that barium directly inhibited the calcium-activated potassium channels. This inhibition was non-competitive and, at a 10 mM barium concentration, about 50% of the channels were blocked. This is in agreement with the electrophysiological studies of Iwatsuki and Petersen (1985), who showed that barium could block calcium-activated
potassium channels in pig pancreas, and are consistent with the present view that barium is a general inhibitor of such channels when used in the millimolar range. It remains to be established on which site of the channel barium acts. It has recently been reported that barium inhibits the release of amylase induced by CCK-8 from permeabilized rat pancreatic acini but not from intact acini (Fuller, Eckhardt and Schulz, 1989). This has been interpreted as an evidence for the presence of calcium-activated potassium channels on the membrane of zymogen granules. The blockade of these channels by barium prevents the entry of potassium (and its counter-anion, chloride) in the granules and is likely to prevent the osmotic swelling of these granules. This finding also suggests that barium cannot cross an intact plasma membrane. On the other hand, barium can enter the chromaffin cells through a channel for divalent cations (Artalejo, Garcia and Aunis, 1987). More recently, it has been reported that barium could enter inside lacrimal glands via the calcium channel activated by agonists (Kwan and Putney, 1990). Similar channels have been described in the parotid gland (Takemura and Putney, 1989), where they are activated by carbamylcholine (Mertz er al., 1990). This could explain why barium is a better inhibitor of calcium-activated potassium channels (see Fig. 2) and of salivary secretion (Young et al., 1987) in the presence of high concentrations of carbamylcholine. It has also been demonstrated that barium could block *6Rb+ influx in inside-out vesicles from kidney cells (Klaerke, Karlish and Jorgensen, 1987). It is thus possible that it blocks the potassium channel after binding to a site located on the cytoplasmic side of the channel. One possibility would be that barium binds to and blocks the calcium-binding site of the channel. Such a hypothesis is consistent with the fact that barium blocks some intracellular calcium-activated enzymes (Warchek et al., 1987). For secretion, the action of barium is opposite to that described above as it can mimic the action of extracellular calcium and stimulate amylase secretion. This is even more obvious in our experiments where extracellular calcium had been chelated with EGTA. Some important conclusions can be drawn from such experiments. First, removal of extracellular calcium did not affect the efflux of 86Rb+, confirming that the efflux of the isotope is strictly dependent (at least in short experiments) on the mobilization of an intracellular pool of calcium (Foskett et al., 1989). Second, the efflux of 45Ca2+ was not modified by the absence of calcium in the extracellular medium, showing that a Ca/Ca exchange is not involved in the extrusion of 45Ca2+. Third, the basal amylase release could be stimulated by the increase in the extracellular calcium concentration. In rat parotid acini, calcium is thus a potent secretagogue by itself and does not require the activation of protein kinase C by diglycerides to stimulate amylase release. Increasing the extracellular calcium concentration also potentiated the secretory response to carbamylcholine. Two explanations can account for this observation: (1) amylase secretion was measured after 20 min, and the acini have thus been exposed to EGTA for this long period, which is likely to deplete intracellular stores of calcium and to decrease the response to the muscarinic agent; (2) it
Barium and rat parotid gland should also be recalled that within 20min, the response to carbamylcholine relies not only on intracellular stores of calcium, but also on extracellular calcium, the influx of which is stimulated by the depletion of the IP,-sensitive intracellular store (Putney, 1986b). It remains that increasing the extracellular concentration of calcium above 1 mM activates amylase secretion. In the absence of free extracellular calcium, barium inhibited the efflux of *‘Rb+ but did not affect the efflux of 4SCa2+.It could still stimulate amylase release, albeit at a lower rate than calcium. Our ‘calcium-free’ medium contained 0.1 mM calcium and 0.5 mM EGTA, and the apparent stimulatory effect of barium might be secondary to the displacement of the calcium bound to EGTA. However, this is unlikely because the effect of high concentrations of barium is greater than the effect observed in the presence of EGTA 0.5 mM and 1.1 mM calcium, which would lead to a free calcium concentration higher than 0.1 mM. In conclusion, barium has two effects in rat parotid acini. It is an antagonist of the calcium-activated potassium channels and an agonist on amylase secretion. Acknowledgement-This
work was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique.
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