Department of Physiology II, University of Saarland, D-66421 Homburg/Saar, Germany
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ABSTRACT |
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We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 ± 0.3 µm/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+ exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 ± 0.6 µm/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di-tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool to the cytosol.
intracellular calcium pools; bafilomycin; exocrine pancreas
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INTRODUCTION |
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RECEPTOR-MEDIATED activation of numerous cell types leads to an increase in the cytosolic inositol 1,4,5-trisphosphate (IP3) concentration and a consequent release of Ca2+ from internal stores. This results in a rise in the cytosolic free Ca2+ concentration ([Ca2+]i) (34). Subsequently, Ca2+ influx from the extracellular space occurs, which has been termed "capacitative" Ca2+ entry (28). In rat and mouse pancreatic acinar cells, hormone-evoked Ca2+ signals start in the luminal cell pole and then spread in the form of Ca2+ waves toward the basolateral side of the cell (14, 15, 27, 38). In many cell types, Ca2+ waves occur either spontaneously or after stimulation by hormones and neurotransmitters or by treatments promoting Ca2+ influx into the cell (7, 9, 10, 15, 21, 35). Ca2+ wave propagation has been explained by sequential Ca2+ release from Ca2+ stores in series (9, 21), and it has been suggested for many cell types that the mechanism underlying Ca2+ wave propagation involves Ca2+-induced Ca2+ release (CICR). Evidence suggests that CICR is also important for the propagation of cytosolic Ca2+ waves in pancreatic acinar cells (21). It has been shown that the shape of Ca2+ waves as well as the speed of wave propagation differ from one cell type to another (13, 35). This indicates that differences in the regulation of Ca2+ waves could play a role in the regulation of different cell functions (7).
In numerous cell types, including pancreatic acinar cells, cytoplasmic organelles have a lower pH than the cytosol (24, 39). This acidity could be important for storage of Ca2+ in these organelles (32). In studies on mouse pancreatic acinar cells, it has been found that a decrease in the acidity inside of zymogen granules inhibits secretagogue-stimulated Ca2+ spikes (39) and that hormone-evoked Ca2+ signals require the existence of subcellular gradients of pH (12).
The effects of changes in the extracellular pH (pHo) on [Ca2+]i and Ca2+ influx have been extensively studied in different cell types (3, 20), and it had been proposed that the intracellular pH (pHi) might be a modulator of Ca2+ signals (42). In Ca2+ stores of pancreatic acinar cells, the presence of a Ca2+/H+ exchanger and an H+ pump in parallel, which creates the driving force for Ca2+ uptake, had been suggested (31). Furthermore, it had been shown that stimulation of pancreatic acinar cells with Ca2+-mobilizing agonists leads to an increase in [Ca2+]i and to an acidification of the cytosol due to Ca2+ extrusion and Ca2+ uptake via Ca2+/H+ exchangers across the plasma membrane and the membrane of intracellular Ca2+ stores, respectively (11). In addition to pHo (20), pHi can also modulate Ca2+ influx (41) and is able to modify secretagogue-evoked Ca2+ oscillations (12, 43). In a study on HT-29 cells, it had been recently proposed that intracellular acidification enhances [Ca2+]i transients (22). These data suggest that, in addition to other factors regulating Ca2+ signals in living cells, there is a close relationship between pHi and [Ca2+]i in different cell systems.
Taking into account the rapid propagation of Ca2+ waves across the cell and the close relationship between pHi and Ca2+ signaling, we wanted to examine the effect of changes in pHi on ACh-induced Ca2+ waves in mouse pancreatic acinar cells. To carry out these investigations, confocal laser scanning microscopy was used to follow local changes in [Ca2+]i in individual mouse pancreatic acinar cells loaded with the fluorescent probe fluo 3.
Our results show that a decrease in cytosolic pH, as well as inhibition of the vacuolar type H+-ATPase present in intracellular organelles, results in slowing of ACh-induced Ca2+ wave propagation. On the other hand, cytosolic alkalinization had no effect on the spreading of hormone-evoked Ca2+ signals. A decrease in the speed of ACh-induced Ca2+ waves by cytosolic acidification could also be observed when Ca2+ reuptake into IP3-sensitive stores was inhibited by 2,5-di-tert-butylhydroquinone (tBHQ). Furthermore, the effect of cytosolic acidification was not additive to inhibition of CICR by ruthenium red. These data therefore suggest that the decrease in the speed of Ca2+ wave propagation could be due to inhibition of Ca2+ release from intracellular stores caused by cytosolic acidification or by a decrease in the proton gradient across intracellular Ca2+ pool membranes. We conclude that cytosolic pH and/or intraluminal pH of Ca2+ stores should be considered an important factor regulating Ca2+ signaling in mouse pancreatic acinar cells.
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EXPERIMENTAL PROCEDURES |
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Animals and chemicals. Adult male CD-1 mice were used for this study. All materials used were obtained from Sigma Chemical, except fluo 3-AM and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (from Molecular Probes), bafilomycin A1 (from Calbiochem-Novabiochem), and tBHQ (from Aldrich Chemical).
Preparation of isolated acinar cells. After death of the animal by cervical dislocation, the pancreas was rapidly removed and placed into a preparation buffer of the following composition (in mM): 130 NaCl, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 1.2 KH2PO4, 10 HEPES, 10 glucose, 0.2% (wt/vol) albumin, and 0.01% (wt/vol) trypsin inhibitor; the pH of this solution was adjusted to 7.4 with NaOH. Adherent blood vessels and fat tissue were removed, and the pancreas was injected with 1 ml of the preparation buffer supplemented with collagenase type V (30 U/ml) and incubated at 37°C in a shaking water bath (200 cycles/min) for 10 min. Enzymatic digestion of the tissue was followed by gently pipetting the cell suspension through tips of decreasing diameter for mechanical dissociation of the cells. The cells were centrifuged for 2 min at 30 g, and the pellet was resuspended in preparation buffer without collagenase. Finally, cells were washed twice in preparation buffer in the absence of collagenase. With this isolation procedure, we obtained single cells as well as small clusters consisting of two to up to five cells.
Cytosolic Ca2+ measurements. For measurement of [Ca2+]i, cells were loaded with 4 µM fluo 3-AM for 30 min at room temperature (22°C) following previously established methods (26). After the dye was loaded, the cells were kept at 4°C and the experiments were performed within the next 4 h. For Ca2+-dependent fluorescence signal monitoring, aliquots of the cell suspension were placed onto polylysine-coated glass coverslips attached to the bottom of a perifusion chamber. The cells were continuously superfused with a NaHEPES buffer (buffer 1) containing (in mM) 140 NaCl, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.4 with NaOH. With the use of a confocal laser-scanning system (Bio-Rad, MRC-600), fluorescence images of 128 × 128 pixels with a resolution of 0.463 µm/pixel were recorded every 0.28 s. Small rectangular areas were selected in the luminal and the basolateral poles of the cells. The difference in time between the increase in [Ca2+]i at both cell regions was determined, and the speed (µm/s) of the ACh-induced Ca2+ signal was calculated. All experiments were performed at room temperature.
pHi measurements. For the measurement of pHi, isolated pancreatic acinar cells were loaded with 5 µM of BCECF-AM for 30 min at room temperature (22°C) (12). After dye was loaded, the cells were kept at 4°C and the experiments were performed within the next 4 h.
For quantification of fluorescence, aliquots of cell suspension were placed onto polylysine-coated coverslips attached to the bottom of a perfusion chamber superfused with NaHEPES and alternatively excited at 440 and 490 nm. Emitted fluorescence was recorded at 515 nm employing a microscopic imaging system (TILL Photonics, Planegg, Germany) coupled to an inverted microscope (Carl Zeiss).Maneuvers for changing cytosolic pH.
Acidification of cells was carried out by reversal of the
Na+/H+
exchanger by perifusion of the cells with a buffer containing N-methyl-D-glucamine
(NMDG) instead of Na+. With this
procedure, pancreatic acinar cells reached pH values of ~6.60 ± 0.04 after 5 min (12). The composition of the NMDG buffer
(buffer 2) was (in mM) 140 NMDG, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose,
pH adjusted to 7.4 with HCl. In these two protocols, cells were
perifused with the modified buffer for 5 min before stimulation with
ACh. A second way to produce cytosolic acidification consisted of a
blockade of the Na+/H+
exchanger with ethylisopropylamiloride (EIPA;
104 M) added to
buffer 1. In previous studies (19),
inhibition of
Na+/H+
exchange was shown to lead to a rapid and reversible acidification of
the cytosol.
Analysis of data. Data show the mean propagation rate of Ca2+ waves (in µm/s ± SE of the mean). Statistical analysis was performed by Student's t-test, and only P values <0.05 were stated as significant.
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RESULTS |
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ACh-evoked Ca2+ signals. As shown in Fig. 1A, stimulation of pancreatic acinar cells with 500 nM ACh resulted in an initial increase in [Ca2+]i at the luminal cell pole and subsequent spreading of the Ca2+ signal. The Ca2+ signal reached the basolateral cell side with a delay of 0.78 ± 0.02 s (n = 108 experiments/321 cells). From the distance between both areas selected at each side of the cell and the difference in time between the increase in the fluorescence in these regions, we could determine the speed of the Ca2+ wave. In the presence of ACh, the mean speed of the Ca2+ wave was 16.1 ± 0.3 µm/s (n = 108 experiments/321 cells; Fig. 1A).
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Effect of cytosolic acidification on ACh-evoked Ca2+ signals. Substitution of Na+ with NMDG in the perifusion medium leads to acidification of the cytosol due to reversal of the Na+/H+ exchanger (12, 19). When cells were stimulated with ACh in a Na+-free solution (buffer 2), Ca2+ wave propagation from the luminal to basolateral cell pole took 1.89 ± 0.12 s and the mean speed of the ACh-evoked Ca2+ waves was 7.5 ± 0.3 µm/s (n = 41 experiments/122 cells). Under these conditions of low pHi, spreading of the ACh-induced Ca2+ wave was much slower compared with the control (16.1 ± 0.3 µm/s; P < 0.001; Fig. 2).
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Effect of ruthenium red on inhibition of CICR. Stimulation of pancreatic acinar cells with 500 nM ACh following 5 min of preincubation in the presence of 100 µM ruthenium red, an inhibitor of CICR (33), led to a slower spreading of the Ca2+ wave, similar to that described previously (27). The mean speed of the Ca2+ wave under ruthenium red treatment was 11.6 ± 0.5 µm/s (Fig. 4) (n = 27 experiments/54 cells), which was statistically different compared with the experiments in which pancreatic acinar cells were stimulated with ACh in the absence of ruthenium red (P < 0.001). The effect of a combination of ruthenium red and NMDG was not additive compared with the effect observed in the presence of NMDG alone (Fig. 4).
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Effect of cytosolic alkalinization on ACh-evoked Ca2+ waves. Cytosolic alkalinization by perifusion of the cells with either a buffer containing 30 mM NH4Cl (12, 29) or a buffer of pH 8.0 (3, 20) or by removal of 15 mM sodium propionate from the perifusing buffer (12, 29) revealed no statistical differences in the spreading of ACh-induced Ca2+ waves compared with control experiments. The mean propagation rates of the Ca2+ waves were 16.2 ± 0.8 µm/s (n = 12 experiments/37 cells) at a pHo = 8.0, 14.6 ± 0.9 µm/s (n = 11 experiments/27 cells) under acute addition of NH4Cl, and 13.6 ± 0.7 µm/s (n = 9 experiments/25 cells) after removal of sodium propionate (Fig. 5).
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Effect of vacuolar-type H+-ATPase inhibition on ACh-evoked Ca2+ waves. In pancreatic acinar cells, as well as in many other cell types, the pH in the lumen of different intracellular organelles is more acidic than in the cytosol (24, 39). It has been proposed that this low pH is maintained by a vacuolar-type H+-ATPase, which is located in the membrane of these organelles and can be specifically inhibited by bafilomycin A1 (4, 47). The ATP-driven H+ gradient could be used for Ca2+ sequestration into Ca2+ stores through a Ca2+/H+ exchanger (11, 31).
Stimulation of the cells with 500 nM ACh after 5 min of preincubation in the presence of 50 nM of the H+-ATPase inhibitor bafilomycin A1 led to a slower spreading of the Ca2+ wave. The mean speed of Ca2+ wave under bafilomycin A1 treatment (see Fig. 2) was 11.1 ± 0.6 µm/s (n = 12 experiments/27 cells), which was statistically different compared with the experiments in which pancreatic acinar cells were stimulated with ACh in the absence of bafilomycin A1 (P < 0.001). The effect of a combination of bafilomycin A1 and NMDG was not additive compared with the effect observed in the presence of NMDG alone (data not shown). As shown in Fig. 6, perifusion of pancreatic acinar cells with a buffer containing 50 nM bafilomycin A1 did not lead to changes in pHi (n = 9 experiments/62 cells). As a control, cells were stimulated with 500 nM ACh at the end of the experiment. This led to a transient acidification of the cytosol, which is due to the release of Ca2+ from intracellular stores (11).
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Effect of endoplasmic reticulum Ca2+-ATPase inhibition on ACh-evoked Ca2+ waves. After an increase in [Ca2+]i following hormonal stimulation, Ca2+ is extruded via the plasma membrane Ca2+-ATPase and sequestered by a Ca2+-ATPase located in the membrane of intracellular stores (6, 16). The latter can be inhibited by low concentrations of Ca2+-ATPase inhibitors such as thapsigargin or tBHQ (18, 25).
When the cells were preincubated for 5 min in the presence of 100 nM tBHQ and then stimulated with ACh, a faster speed of the Ca2+ wave compared with the control was observed. The mean speed of ACh-induced Ca2+ waves in the presence of tBHQ was 25.6 ± 1.8 µm/s (n = 12 experiments/34 cells) (Fig. 7). Figure 1C shows a typical trace of fluorescence signals both at the luminal and the basolateral cell poles following stimulation of pancreatic acinar cells with 500 nM ACh in the presence of 100 nM tBHQ. The fluorescence first increased at the luminal pole of the cell and then spread toward the basolateral pole. The mean speed of ACh-induced Ca2+ waves in the presence of tBHQ was higher than in control cells (P < 0.001) (see Fig. 7).
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ACh-evoked signals in Ca2+-free medium. We considered a contribution of extracellular Ca2+ to the speed of the Ca2+ wave propagation and tested the possibility that the effect of intracellular acidification on Ca2+ wave spreading could be due at least in part to an inhibition of the Ca2+ influx. We therefore performed experiments in Ca2+-free medium in the presence of 1 mM EGTA. Table 1 shows the mean speed of ACh-induced Ca2+ waves in mouse pancreatic acinar cells under different conditions, both in Ca2+-containing and Ca2+-free media. Except in the experiments performed in the presence of tBHQ, no statistical differences were found in the experiments carried out in Ca2+-free medium compared with those in the presence of Ca2+ in the perifusion medium.
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DISCUSSION |
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We have used fluo 3-loaded mouse pancreatic acinar cells and a confocal laser scanning microscope to investigate the relationship between Ca2+ signals evoked by supramaximal concentrations of ACh and the cytosolic pH. Our experiments show that, in accordance with data obtained by others on rat and mouse pancreatic acinar cells (14, 15, 21, 38, 46), ACh-induced Ca2+ signals originate in the luminal cell pole, from where they spread to the basal cell membrane in the form of a Ca2+ wave. Our measurements suggest that the speed of ACh-evoked Ca2+ waves is influenced by a decrease in the pHi. Under control conditions, Ca2+ wave propagation was 16.1 ± 0.3 µm/s. The velocity of the Ca2+ waves significantly decreased when the acinar cells were acidified before stimulation with ACh. In contrast, cytosolic alkalinization had no effect on the spreading speed of ACh-induced Ca2+ waves.
Ca2+ release from Ca2+ pools. Previous studies on pancreatic acinar cells gave evidence for different Ca2+ pools with distinct properties in Ca2+ release and Ca2+ reuptake (25, 31, 36). It has been shown that IP3 applied via a patch pipette led to an initial increase in the [Ca2+]i in the luminal cell pole, even when the patch pipette was attached to the basal cell membrane (38). This observation led to a model in which highly IP3-sensitive Ca2+ stores were located in the luminal cell pole. Recently, we could show that in mouse pancreatic acinar cells propagation of hormone-evoked Ca2+ waves involves different Ca2+ release mechanisms. After initial Ca2+ release from the highly IP3-sensitive luminal Ca2+ pool, spreading of the Ca2+ signal to the basal cell pole is facilitated by a protein kinase C (PKC)-sensitive Ca2+ release mechanism from secondary Ca2+ pools located in series between the luminal and basal pole of pancreatic acinar cells (27). Activation of PKC leads to an inhibition of Ca2+ release from the secondary stores and therefore slows down spreading of hormone-evoked Ca2+ waves. Ca2+ release from secondary pools is presumably triggered by a Ca2+-induced Ca2+ release mechanism and in addition could also involve IP3 receptors of low affinity.
Ca2+ uptake into Ca2+ pools. Ca2+ uptake into IP3-sensitive Ca2+ pools occurs via thapsigargin-sensitive Ca2+ transports, which belong to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) family (16, 18, 25). However, another model for Ca2+ uptake, which involves a Ca2+/H+ exchanger and a vacuolar "V-type" H+-ATPase in parallel, has also been suggested to operate in IP3-sensitive Ca2+ pools (36, 37). Abolishment of H+ gradients across vacuolar membranes of Trypanosoma by use of protonophores as well as inhibition of the H+ pump resulted in an inhibition of Ca2+ uptake (8, 32, 44). In pancreatic acinar cells and insulin-secreting cells, disruption of subcellular H+ gradients by application of protonophores or H+-pump inhibitors led to depletion of intracellular Ca2+ pools and consequently impaired subsequent Ca2+ mobilization in response to hormones or IP3 (2, 12, 36). This indicates the presence of acidic Ca2+ pools in acinar cells and the requirement of subcellular H+ gradients for Ca2+ uptake into these Ca2+ pools (12, 36, 37).
Effect of intracellular acidification on Ca2+ wave propagation. In the present study, we have demonstrated that the propagation rate of hormone-induced Ca2+ waves in mouse pancreatic acinar cells is slowed down by intracellular acidification. This observation can be explained by pH-dependent reduction in Ca2+ release and/or by pH-dependent augmentation of Ca2+ uptake into intracellular stores during Ca2+ wave spreading. Because Ca2+ uptake into intracellular stores is partially driven by an H+ gradient (11, 31, 37), abolishment or reduction of this gradient by cytosolic acidification or application of bafilomycin A1, an inhibitor of V-type H+-ATPase, should reduce Ca2+ reuptake during spreading of the Ca2+ signal and, therefore, accelerate Ca2+ wave propagation. However, both cytosolic acidification with various methods as well as inhibition of the V-type H+-ATPases with bafilomycin A1 led to a reduction in the propagation rate of Ca2+ waves. This makes it unlikely that the pH-dependent reduction in the speed of ACh-induced Ca2+ waves is due to an impaired Ca2+ reuptake. Furthermore, we have shown that inhibition of Ca2+ uptake by tBHQ leads to an increase in the speed of Ca2+ wave propagation (Ref. 27 and Fig. 7). If cytosolic acidification would inhibit Ca2+ uptake, it should not have any effect in the presence of tBHQ. As shown in Fig. 7, however, both NMDG and bafilomycin A1 decreased the speed of Ca2+ waves to the level seen also in the absence of tBHQ (compare Figs. 2 and 7). Therefore, it is more likely that cytosolic acidification, similar to PKC activation (27), leads to a reduction in Ca2+ release from secondary stores and therefore decreases the propagation rate of cytosolic Ca2+ waves.
Furthermore, the effects of ruthenium red, an inhibitor of CICR in skeletal muscle (33), and a voltage-dependent Ca2+ channel in pancreatic endoplasmic reticulum (30) reduced ACh-evoked Ca2+ wave propagation to the same extent as cytosolic acidification and the effects of both were not additive (Fig. 4). This suggests that acidification, similarly to ruthenium red, inhibits CICR and therefore reduces the speed of ACh-induced Ca2+ waves. On the other hand, it is known that following stimulation of the cells a Ca2+ entry pathway is activated. The entry of Ca2+ from the extracellular space could play a role in the propagation of Ca2+ signals in our conditions, as proposed previously (10). It has also been suggested that intracellular acidification stimulates Ca2+ influx in LLC-PK1 cells (5). In contrast, other studies carried out on HT-29 cells revealed that Ca2+ influx is decreased at acidic pHi (22). Our experiments performed in the absence of extracellular Ca2+ show that, in pancreatic acinar cells, spreading of ACh-induced Ca2+ waves is not different in the absence compared with the presence of extracellular Ca2+. It therefore seems unlikely that cytosolic acidification would lead to a decrease in Ca2+ influx and therefore to a slower propagation of ACh-induced signals. This is in accordance with the results shown in several studies performed on smooth muscle cells (1) as well as on rat pancreatic acinar cells (12), in which Ca2+ influx was shown to be not affected by intracellular acidification. Furthermore, the possibility that pHi might affect the Ca2+ buffering capacity of the cytosol has to be considered. Competition between Ca2+ and H+ for binding sites would lead to a higher free [Ca2+]i under acidic conditions that consequently could lead to a higher Ca2+ release from internal Ca2+ stores by CICR mechanisms and subsequent faster spreading of the Ca2+ signals. However, our results show that spreading of Ca2+ waves was slower under acidic conditions. Furthermore, we did not see changes in the baseline fluorescence under acidifying or alkalinizing conditions. Another possibility to explain the effect of acidic pHi on Ca2+ wave propagation could be the inhibition of IP3 formation and a consequent decrease in IP3-dependent Ca2+ release. However, in studies on HT-29 cells, carbachol-induced IP3 formation was shown not to change in response to cytosolic acidification with weak acids, whereas alkalinization augmented carbachol-induced IP3 formation (23). Because in our experiments acidic pHi but not alkaline pHi had an effect on the speed of Ca2+ waves, it seems unlikely that the lower rate of Ca2+ wave propagation could be explained by pH-dependent changes in the production of IP3. In former studies (40, 45), binding of IP3 to its receptor was shown to be pH dependent. Acidification led to a diminished IP3-induced Ca2+ release, whereas alkalinization increased IP3 sensitivity of the Ca2+ stores. Here, we could show that cytosolic alkalinization did not lead to a faster spreading of the Ca2+ signals. Furthermore, a reduction in the propagation rate of ACh-evoked Ca2+ waves could be observed by cytosolic acidification as well as by application of bafilomycin A1. Because bafilomycin A1 had no effect on the cytosolic pH (Fig. 6), it is unlikely that a pH-dependent decrease in IP3 binding can explain the reduced propagation rate of Ca2+ waves. Inhibition of V-type H+-ATPase by bafilomycin A1 should lead to an increase in the intraluminal pH of compartments containing this transport mechanism. We therefore suggest that the intraluminal pH of Ca2+ stores and/or a decrease of the pH gradient across the membrane of the Ca2+ store could also play a regulatory role in the control of Ca2+ release. It has been suggested by Minta et al. (17) that careful attention should be paid when working with fluo 3, as this dye could show changes in fluorescence properties dependent on pH. However, in the pH range of our experiments, any changes in the fluorescence of this Ca2+ probe could not be observed under basal or stimulated conditions. Together, we conclude from our data that cytosolic acidification, intraluminal alkalinization, and/or a decrease in the H+ gradient directed from the Ca2+ stores to the cytosol leads to a decrease in CICR from Ca2+ pools involved in hormone-induced Ca2+ wave propagation. As a consequence, the speed of the Ca2+ wave in response to ACh, which is initiated in the luminal cell pole, is decreased. ![]() |
ACKNOWLEDGEMENTS |
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We thank the Deutsche Forschungsgemeinschaft (SFB 246, B11/A9), Consejería de Educación y Juventud-Junta de Extremadura (PRI96100010), and The Wellcome Trust for support of this study.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address reprint requests to I. Schulz.
Received 20 January 1998; accepted in final form 2 June 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Batlle, D. C.,
R. Peces,
M. S. LaPointe,
M. Ye,
and
J. T. Daugirdas.
Cytosolic free calcium regulation in response to acute changes in intracellular pH in vascular smooth muscle.
Am. J. Physiol.
264 (Cell Physiol. 33):
C932-C943,
1993
2.
Bode, H.-P.,
A. Himmen,
and
B. Göke.
Evidence for vacuolar-type proton pumps in nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores of insulin-secreting cells.
Pflügers Arch.
432:
97-104,
1996[Medline].
3.
Borle, A. B.,
and
C. Bender.
Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na+-Ca2+ and Na+-H+ antiporter interactions.
Am. J. Physiol.
261 (Cell Physiol. 30):
C482-C489,
1991
4.
Bowman, E. J.,
A. Siebers,
and
K. Altendorf.
Bafilomycins: a classs of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells.
Proc. Natl. Acad. Sci. USA
85:
7972-7976,
1988[Abstract].
5.
Burns, K. D.,
T. Homma,
M. D. Breyer,
and
R. C. Harris.
Cytosolic acidification stimulates a calcium influx that activates Na+/H+ exchange in LL-PK1.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F617-F625,
1991
6.
Camello, P. J.,
J. Gardner,
O. H. Petersen,
and
A. V. Tepikin.
Calcium dependence of calcium extrusion and calcium uptake in mouse pancreatic acinar cells.
J. Physiol. (Lond.)
490:
585-593,
1996[Abstract].
7.
Clapham, D. E.,
and
J. Sneyd.
Intracellular calcium waves.
Adv. Second Messenger Phosphoprotein Res.
30:
1-24,
1995[Medline].
8.
Docampo, R.,
D. A. Scott,
A. E. Vercesi,
and
S. N. J. Moreno.
Intracellular Ca2+ storage in acidocalciosomes of Trypanosoma cruzi.
Biochem. J.
310:
1005-1012,
1995[Medline].
9.
Dupont, G.,
and
A. Goldbeter.
Properties of intracellular Ca2+ waves generated by a model based on Ca2+-induced Ca2+ release.
Biophys. J.
67:
2191-2204,
1994[Abstract].
10.
Girard, S.,
and
D. Clapham.
Acceleration of intracellular calcium waves in Xenopus oocytes by calcium influx.
Science
260:
229-232,
1993[Medline].
11.
González, A.,
P. J. Camello,
J. A. Pariente,
and
G. M. Salido.
Free cytosolic calcium levels modify intracellular pH in rat pancreatic acini.
Biochem. Biophys. Res. Commun.
230:
652-656,
1997[Medline].
12.
González, A.,
J. A. Pariente,
G. M. Salido,
and
P. J. Camello.
Intracellular pH and calcium signalling in rat pancreatic acinar cells.
Pflügers Arch.
434:
609-614,
1997[Medline].
13.
Jafri, M. S.,
and
J. Keizer.
Diffusion of inositol 1,4,5-trisphosphate but not Ca2+ is necessary for a class of inositol 1,4,5-trisphosphate-induced Ca2+ waves.
Proc. Natl. Acad. Sci. USA
91:
9485-9589,
1994
14.
Kasai, H.,
and
G. J. Augustine.
Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas.
Nature
348:
735-738,
1990[Medline].
15.
Kasai, H.,
Y. X. Li,
and
Y. Miyashita.
Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas.
Cell
74:
669-677,
1993[Medline].
16.
Lytton, J.,
M. Westlin,
S. E. Burk,
G. E. Shull,
and
D. H. MacLennan.
Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.
J. Biol. Chem.
267:
14483-14489,
1992
17.
Minta, A.,
J. P. Y. Kao,
and
R. Y. Tsien.
Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.
J. Biol. Chem.
264:
8171-8178,
1989
18.
Moore, G. A.,
D. J. McConkey,
G. E. N. Kass,
P. J. O'Brien,
and
S. Orrenius.
2,5-Di(tert-butyl)-1,4-benzohydroquinone, a novel inhibitor of liver microsomal Ca2+ sequestration.
FEBS Lett.
224:
331-336,
1987[Medline].
19.
Muallem, S.,
and
P. A. Loessberg.
Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3 transporters by Ca2+-mobilizing agonists.
J. Biol. Chem.
265:
12813-12819,
1990
20.
Muallem, S.,
S. J. Pandol,
and
T. G. Beeker.
Modulation of agonist-activated calcium influx by entracellular pH in rat pancreatic acini.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G917-G924,
1989
21.
Nathanson, M. H.,
P. J. Padfield,
A. J. O'Sullivan,
A. D. Burgstahler,
and
J. D. Jamieson.
Mechanism of Ca2+ wave propagation in pancreatic acinar cells.
J. Biol. Chem.
267:
18118-18121,
1992
22.
Nitschke, R.,
N. Benning,
S. Ricken,
J. Leipziger,
K. G. Fischer,
and
R. Greger.
Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells.
Pflügers Arch.
434:
466-474,
1997[Medline].
23.
Nitschke, R.,
A. Riedel,
S. Ricken,
J. Leipziger,
N. Benning,
K. G. Fisher,
and
R. Greger.
The effect of intracellular pH on cytosolic Ca2+ in HT29 cells.
Pflügers Arch.
433:
98-108,
1996[Medline].
24.
Orci, L.,
M. Ravazzola,
and
R. W. G. Anderson.
The condensating vacuole of exocrine cells is more acidic than the mature secretory vesicle.
Nature
326:
77-79,
1987[Medline].
25.
Ozawa, T.,
F. Thévenod,
and
I. Schulz.
Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells.
J. Membr. Biol.
144:
111-120,
1995[Medline].
26.
Pfeiffer, F.,
A. Schmid,
and
I. Schulz.
Capacitative Ca2+ influx and a Ca2+ dependent nonselective release mechanism cation pathway are discriminated by genistein in mouse pancreatic acinar cells.
Pflügers Arch.
430:
916-922,
1995[Medline].
27.
Pfeiffer, F.,
L. Sternfeld,
A. Schmid,
and
I. Schulz.
Control of Ca2+ wave propagation in mouse pancreatic acinar cells.
Am. J. Physiol.
274 (Cell Physiol. 43):
C663-C672,
1998
28.
Putney, J. W.
A model for receptor-regulated calcium entry.
Cell Calcium
7:
1-12,
1988.
29.
Roos, A.,
and
W. F. Boron.
Intracellular pH.
Physiol. Rev.
61:
296-434,
1981
30.
Schmid, A.,
M. Dehlinger-Kremer,
I. Schulz,
and
H. Gögelein.
Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.
Nature
346:
374-376,
1990[Medline].
31.
Schulz, I.,
F. Thevenod,
and
M. Dehlinger-Kremer.
Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitocondrial Ca2+ pools.
Cell Calcium
10:
325-336,
1989[Medline].
32.
Scott, D. A.,
S. N. J. Moreno,
and
R. Docampo.
Ca2+ storage in Trypanosoma brucei: the influence of cytoplasmic pH and importance of vacuolar acidity.
Biochem. J.
310:
789-794,
1995[Medline].
33.
Smith, J. S.,
R. Coronado,
and
G. Meissner.
Single-channel calcium and barium currents of large and small conductance for sarcoplasmic reticulum.
Biophys. J.
50:
921-928,
1986[Abstract].
34.
Streb, H.,
R. F. Irvine,
M. J. Berridge,
and
I. Schulz.
Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate.
Nature
306:
67-69,
1983[Medline].
35.
Takamatsu, T.,
and
B. Wier.
Calcium waves in mammalian heart: quantification of origin, magnitude, waveform and velocity.
FASEB J.
4:
1519-1525,
1990
36.
Thévenod, F.,
M. Dehlinger-Kremer,
T. P. Kemmer,
A. L. Christian,
B. V. L. Potter,
and
I. Schulz.
Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and -insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells.
J. Membr. Biol.
109:
173-186,
1989[Medline].
37.
Thévenod, F.,
and
I. Schulz.
H+-dependent calcium uptake into an IP3-sensitive calcium pool from rat parotid gland.
Am. J. Physiol.
255 (Gastrointest. Liver Physiol. 18):
G429-G440,
1988
38.
Thorn, P.,
A. L. Lawrie,
P. M. Smith,
D. V. Gallacher,
and
O. H. Petersen.
Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate.
Cell
74:
661-668,
1993[Medline].
39.
Titievsky, A. V.,
T. Takao,
and
A. V. Tepikin.
Decrease in acidity inside zymogen granules inhibits acetylcholine- or inositol trisphosphate-evoked cytosolic Ca2+ spiking in pancreatic acinar cells.
Pflügers Arch.
432:
938-940,
1996[Medline].
40.
Tsukioka, M.,
M. Iino,
and
M. Endo.
pH dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in permeabilized smooth muscle cells of the guinea-pig.
J. Physiol. (Lond.)
475:
369-375,
1994[Abstract].
41.
Tsunoda, Y.
Cytosolic free calcium spiking affected by intracellular pH change.
Exp. Cell Res.
188:
294-301,
1990[Medline].
42.
Tsunoda, Y.
Receptor-operated Ca2+ signaling and crosstalk in stimulus-secretion coupling.
Biochim. Biophys. Acta
1154:
105-156,
1993[Medline].
43.
Tsunoda, Y.,
H. Yoshida,
and
C. Owyang.
Intracellular control of IP3-independent Ca2+ oscillations in pancreatic acini.
Biochem. Biophys. Res. Commun.
222:
265-272,
1996[Medline].
44.
Vercesi, A. E.,
S. N. J. Moreno,
and
R. Docampo.
Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei.
Biochem. J.
304:
227-233,
1994[Medline].
45.
Worley, P. F.,
J. M. Baraban,
S. Supattapone,
V. S. Wilson,
and
S. H. Snyder.
Characterization of inositol trisphosphate receptor in brain. Regulation by pH and calcium.
J. Biol. Chem.
262:
12132-12136,
1987
46.
Xu, X.,
W. Zeng,
J. Diaz,
and
S. Muallem.
Spacial compartmentation of Ca2+ signaling complexes in pancreatic acini.
J. Biol. Chem.
271:
24684-24690,
1996
47.
Yoshimori, T.,
A. Yamamoto,
Y. Moriyama,
M. Futai,
and
Y. Tashiro.
Bafilomycin A1: a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.
J. Biol. Chem.
15:
17707-17712,
1991.