Isoproterenol potentiates alpha -adrenergic and muscarinic receptor-mediated Ca2+ response in rat parotid cells

Akihiko Tanimura, Akihiro Nezu, Yosuke Tojyo, and Yoshito Matsumoto

Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of the cAMP pathway on the Ca2+ response elicited by phospholipase C-coupled receptor stimulations were studied in rat parotid cells. Although 1 µM isoproterenol (Iso) itself had no effect on the cytosolic Ca2+ concentration, the pretreatment with Iso potentiated Ca2+ responses evoked by phenylephrine. The potentiating effect of Iso was attributed to a shifting of the concentration-response curves of phenylephrine to the left and an increase in the maximal response. Half-maximal potentiation occurred at 3 nM Iso. Iso also potentiated the Ca2+ response elicited by carbachol. The potentiating effect of Iso was mimicked by forskolin (10 µM) and dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) and was blocked by 10 µM H-89. Iso potentiated the phenylephrine-induced Ca2+ response in the absence of extracellular Ca2+, but Iso did not increase the inositol trisphosphate (IP3) production induced by phenylephrine. These results suggest that the potentiation of the Ca2+ response can be attributed to a sensitization of IP3 receptors by cAMP-dependent protein kinase.

beta -adrenergic receptor; adenosine 3',5'-cyclic monophosphate; calcium release; potentiation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SECRETORY EVENTS IN PAROTID acinar cells are controlled by two intracellular messengers: Ca2+ and cAMP (2, 21). Ca2+ is the principal mediator for fluid secretion and is regulated by the phospholipase C (PLC) pathway, which induces Ca2+ release from intracellular Ca2+ stores through the formation of inositol trisphosphate (IP3). The cAMP pathway is regulated by beta -adrenergic agonists and vasoactive intestinal peptide (VIP) in parotid cells. This pathway promotes amylase exocytosis through the activation of cAMP-dependent protein kinase (PKA) (22, 27).

The involvement of beta -adrenergic receptors in the regulation of cytosolic Ca2+ concentration ([Ca2+]i) has been the subject of considerable debate; however, at present it is generally accepted that the physiological level of beta -adrenergic stimulation does not change the [Ca2+]i in parotid acinar cells (15, 28, 32). Although some earlier studies reported that high concentrations of a beta -adrenergic agonist [>10 µM isoproterenol (Iso)] can increase [Ca2+]i (1, 14), it has been found that these concentrations of Iso stimulate alpha -adrenergic receptors, by which [Ca2+]i is increased (15, 28). It has also been demonstrated that a direct application of cAMP does not induce the Ca2+ release in permeabilized parotid acinar cells (32). These studies have indicated that the cAMP pathway by itself does not induce a Ca2+ response.

Conversely, several studies have suggested that the cAMP pathway is involved in regulating salivary fluid secretion. It has been shown that VIP enhances the fluid secretion elicited by PLC-coupled receptor agonists, such as substance P, ACh, and phenylephrine (4, 7). These enhancing effects of VIP are mimicked by forskolin (17). These observations imply that the cAMP pathway modulates the PLC pathway.

In many cell types, the agonist-induced Ca2+ responses are modulated by the cAMP pathway; the Ca2+ response is either potentiated (5, 6, 10, 13) or inhibited (30, 34) by the cAMP pathway. The present study examined the interaction between the cAMP and PLC pathways in regulating [Ca2+]i in rat parotid cells. It demonstrates that the physiological level of beta -adrenergic stimulation potentiates the [Ca2+]i elevation elicited by phenylephrine or carbachol. The results suggest that the potentiation of the Ca2+ response can be attributed to the enhancement of Ca2+ release by sensitizing the IP3-sensitive Ca2+ stores in parotid cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents. Phenylephrine, carbachol, atropine, Iso, propranolol, forskolin, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), collagenase (type II), trypsin (type III), trypsin inhibitor (type II-S), and BSA were obtained from Sigma (St. Louis, MO). The myo-[2-3H]inositol was purchased from Muromachi Kagaku Kogyo (Tokyo, Japan). Fura 2-AM from Dojin Chemicals (Kumamoto, Japan), phentolamine from Ciba-Geigy (Hyogo, Japan), H-89 from Seikagaku (Tokyo, Japan), and K-252a from Kyowa Medex (Tokyo, Japan) were used. All other reagents were of analytical grade.

Cell preparation. Male Wistar strain rats (2-3 mo) were anesthetized with diethyl ether and killed by cardiac puncture. Parotid glands were minced and digested with trypsin and collagenase as described elsewhere (28). The parotid acinar cells were washed and resuspended in Hanks' balanced salt solution buffered with 20 mM HEPES (pH 7.4) and containing 0.2% BSA (HBSS-HB).

Measurement of [Ca2+]i. Parotid cells were incubated for 30 min with 2 µM fura 2-AM in HBSS-HB at room temperature. The fura 2-loaded cells were washed twice, resuspended in fresh HBSS-HB, and stored at room temperature until use. Fura 2 fluorescence was measured at 37°C with a Hitachi F2000 spectrofluorometer (Hitachi, Tokyo, Japan) with excitation at 340 and 380 nm and emission at 510 nm. The [Ca2+]i was calculated from the fluorescence ratio as described by Grynkiewicz et al. (11).

Measurement of inositol phosphate metabolism. Inositol phosphates were measured using a method described elsewhere (29). Briefly, parotid cells were labeled by incubation with myo-[2-3H]inositol (1.9 MBq/ml) for 100 min at 37°C, washed twice, and resuspended in fresh HBSS-HB containing 1% BSA. The labeled cells were preincubated for 5 min with 10 mM LiCl and then stimulated with either phenylephrine, Iso, or a combination of phenylephrine and Iso at 37°C. The reactions were stopped by addition of HClO4 (final concentration 4.5%). A portion of the extract was then neutralized with 0.5 M KOH-9 mM Na2B4O7. The labeled inositol phosphates were separated using a Bio-Rad AG1-X8 column using the method of Berridge et al. (3).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Adrenergic stimulation potentiates the phenylephrine-induced [Ca2+]i elevation. Figure 1 shows the increase in [Ca2+]i evoked by phenylephrine (0.3-10 µM) in the presence (trace 1) or absence (trace 2) of 1 µM Iso. Iso (1 µM) itself did not alter the basal [Ca2+]i, but the pretreatment with Iso potentiated the Ca2+ response elicited by phenylephrine (Fig. 1, A-C). Application of 0.3 µM phenylephrine did not stimulate the Ca2+ response in the control cells (Fig. 1A, trace 2), whereas it evoked a biphasic Ca2+ response in Iso-treated cells (Fig. 1A, trace 1), indicating that the pretreatment with 1 µM Iso lowered the threshold for the Ca2+ response. Stimulation with 1 and 10 µM phenylephrine induced a biphasic Ca2+ response, and the extent of the peak and the sustained [Ca2+]i elevation were increased in the presence of 1 µM Iso (Fig. 1, B and C). Figure 1D shows the extent of the peak [Ca2+]i elevation after addition of various concentrations of phenylephrine in the presence and the absence of 1 µM Iso. Iso shifted the concentration-effect relationship of phenylephrine to the left and elevated the maximum increase in [Ca2+]i.


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Fig. 1.   Effect of isoproterenol (Iso) on phenylephrine-induced Ca2+ response. A-C: parotid cells were stimulated with 0.3 µM (A), 1 µM (B), or 10 µM (C) phenylephrine (PhL) in presence (trace 1) and absence (trace 2) of 1 µM Iso. Iso or phenylephrine was added to fura 2-loaded parotid cells as indicated by black arrowheads (trace 1) or gray arrowheads (trace 2). D: increase in cytosolic Ca2+ concentration ([Ca2+]i) above basal level measured 10 s after stimulation with various concentrations of phenylephrine in presence () or absence (open circle ) of 1 µM Iso. Values are means ± SE of 4 independent experiments.

The potentiating effect was also observed when Iso was applied after phenylephrine stimulation (Fig. 2A). After the sustained [Ca2+]i elevation with 1 µM phenylephrine, the application of 100 nM Iso increased the [Ca2+]i to a higher sustained [Ca2+]i level within 1 min. The Iso-induced increase in [Ca2+]i after the phenylephrine stimulation was blocked by propranolol, a beta -adrenergic antagonist, confirming that the effect of Iso is mediated by beta -adrenergic receptors. The effects of various concentrations of Iso in elevating the [Ca2+]i above the phenylephrine-induced sustained [Ca2+]i is demonstrated by Fig. 2B. Half-maximal potentiation of the Ca2+ response was attained at an Iso concentration of ~3 nM. To examine whether the potentiating effect of Iso specifically acts on the alpha -adrenergic receptors, the effect of Iso on the carbachol-induced Ca2+ response was examined. Figure 2C shows that the 100 nM carbachol-induced sustained [Ca2+]i was also potentiated after application of 100 nM Iso, suggesting that Iso commonly potentiates the effect of PLC-coupled receptor agonists on Ca2+ responses.


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Fig. 2.   Effect of Iso on sustained [Ca2+]i after stimulation with phenylephrine. A: for trace 1, 1 µM phenylephrine, 100 nM Iso, 100 nM propranolol (Pro), and 100 nM phentolamine (Pt) were added as indicated. Trace 2 is apparent [Ca2+]i in unstimulated parotid cells. B: Iso-dependent increase above 1 µM phenylephrine-induced sustained [Ca2+]i measured 60 s after application of various concentrations of Iso (). Apparent changes in [Ca2+]i in absence of phenylephrine were also determined by 60-s incubation with or without 1 µM Iso (open circle ). Values are means ± SE of 4 independent experiments. C: in trace 1, 100 nM carbachol (CCh), 100 nM Iso, 100 nM propranolol, and 10 nM atropine (Atr) were added as indicated. Trace 2 is apparent [Ca2+]i in unstimulated parotid cells.

Potentiation of phenylephrine-induced [Ca2+]i is mediated by PKA. Next the effect of the PKA inhibitor H-89 on the Iso-induced potentiation was examined. Application of 10 µM H-89 had little or no effect on the sustained increase in [Ca2+]i elicited by 1 µM phenylephrine, but it blocked a subsequent [Ca2+]i elevation by 100 nM Iso almost completely (Fig. 3A). When [Ca2+]i was elevated with 1 µM phenylephrine and 100 nM Iso, the additional application of H-89 reduced the [Ca2+]i to the level before the application of Iso (Fig. 3B). A similar inhibition of the Iso-induced potentiation was observed with 10 µM K-252a, another protein kinase inhibitor (n = 4; data not shown).


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Fig. 3.   Effect of H-89 on Iso-induced potentiation of Ca2+ response. As indicated, 1 µM phenylephrine, 10 µM H-89, and 100 nM Iso were added. A: H-89 added before Iso. B: Iso added before H-89. Results shown are typical representations of 4 independent experiments.

In addition, we examined the effect of an adenylate cyclase activator, forskolin, and the cell-permeant cAMP analog DBcAMP on the [Ca2+]i. Forskolin potentiated the phenylephrine-induced [Ca2+]i elevation, although forskolin itself did not change the basal [Ca2+]i (Fig. 4A). The increases in [Ca2+]i with phenylephrine (1 µM) alone and with phenylephrine plus a 1-min pretreatment with 10 µM forskolin were 26.1 ± 7.9 and 75.8 ± 11.2 nM (means ± SE, n = 4), respectively (above the basal level). When [Ca2+]i was elevated with 1 µM phenylephrine, the subsequent application of 10 µM forskolin caused an additional increase in [Ca2+]i (Fig. 4A, trace 2). The phenylephrine-induced [Ca2+]i elevation was also potentiated by DBcAMP (Fig. 4B). The increases in [Ca2+]i with phenylephrine (1 µM) alone and with phenylephrine plus a 5-min pretreatment with 2 mM DBcAMP were 26.6 ± 7.7 and 52.1 ± 9.1 nM (means ± SE, n = 3), respectively. This potentiated [Ca2+]i elevation was attenuated by 10 µM H-89. These results strongly suggest that the potentiation of the phenylephrine-induced Ca2+ response by Iso is mediated by PKA.


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Fig. 4.   Effect of cAMP-elevating agents on Ca2+ response. Black arrowheads are for trace 1, and gray arrowheads are for trace 2. A: as indicated, 10 µM forskolin (F) and 1 µM phenylephrine were added. B: as indicated, 1 µM phenylephrine and 10 µM H-89 were added after 5-min incubation with (trace 1) or without (trace 2) 2 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Results shown are typical representations of 4 independent experiments.

Iso potentiates Ca2+ release from intracellular stores. To determine whether Iso potentiates the phenylephrine-induced Ca2+ release from intracellular Ca2+ stores, Ca2+ responses were examined in the absence of extracellular Ca2+ (Fig. 5). In Ca2+-free medium containing 0.2 mM EGTA, the increases in [Ca2+]i with phenylephrine (1 µM) alone and with phenylephrine plus a 1-min pretreatment with 100 nM Iso were 19.0 ± 5.4 and 34.0 ± 7.7 nM (means ± SE, n = 4), respectively. Furthermore, when 100 nM Iso was added after the transient increase in [Ca2+]i elicited by 1 µM phenylephrine, Iso evoked an additional transient response in the [Ca2+]i (Fig. 5, trace 2). These results clearly demonstrate that Iso induces Ca2+ release from intracellular Ca2+ stores by potentiating the phenylephrine-coupled signaling pathway.


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Fig. 5.   Ca2+ response induced by phenylephrine and Iso in absence of extracellular Ca2+. As indicated, 0.2 mM EGTA (E), 100 nM Iso, and 1 µM phenylephrine were added to cells in Ca2+-free HBSS-HB. Black arrowheads are for trace 1, and gray arrowheads are for trace 2. Results shown are typical representations of 4 independent experiments.

As shown in Fig. 6, it was verified that Iso has no effect on the Ca2+ response elicited by thapsigargin, an inhibitor of microsomal Ca2+ pumps. In the absence of extracellular Ca2+, 0.5 µM thapsigargin induced a transient increase in [Ca2+]i by leakage of Ca2+ from intracellular Ca2+ stores, and the subsequent addition of CaCl2 (2 mM) resulted in a sustained increase in [Ca2+]i by Ca2+ entry through store-operated Ca2+ channels. Neither of these thapsigargin-dependent Ca2+ responses are altered by Iso. It was also observed that 10 mM caffeine, an activator of ryanodine-sensitive Ca2+ channels, did not induce any detectable change in [Ca2+]i in the presence or absence of Iso (data not shown). These observations suggest that Iso does not potentiate Ca2+ responses other than the Ca2+ release from IP3-sensitive Ca2+ stores.


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Fig. 6.   Effect of Iso on thapsigargin-induced Ca2+ response. As indicated, 0.2 mM EGTA, 100 nM Iso, 0.5 µM thapsigargin (ThG), and 2 mM CaCl2 were added to cells in Ca2+-free HBSS-HB. Black arrowheads are for trace 1, and gray arrowheads are for trace 2. Results shown are typical representations of 5 independent experiments.

Effect of Iso on the phenylephrine-induced inositol phosphate production. The above data indicate that Iso potentiates IP3-dependent Ca2+ release. We further examined whether this potentiation can be attributed to the enhancement of IP3 production. The changes for phenylephrine-induced accumulation of inositol phosphates are shown in Fig. 7A, where the maximum accumulation of IP3 was 10 min after stimulation with 1 µM phenylephrine in the presence of 100 nM Iso and also in its absence. In Fig. 7B, the accumulation of inositol phosphates is represented as a percent increase above the basal value (before stimulation). In this experiment, the basal values of inositol monophosphate (IP1), inositol bisphosphate (IP2), and IP3 were 22,778 ± 3,151, 6,151 ± 1,643, and 717 ± 74 dpm/ml (n = 6), respectively. After 10 min of incubation with 1 and 10 µM phenylephrine, IP3 increased ~1.7-fold and 3.7-fold, respectively. These phenylephrine-induced accumulations of IP3 were not enhanced by 100 nM Iso. During the 10-min incubation with phenylephrine, IP1 and IP2 increased slightly (1.3- to 1.7-fold), whereas there was no enhancement by 100 nM Iso. These data suggest that the Iso-induced potentiation of the Ca2+ response cannot be attributed to the enhancement of the IP3 production.


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Fig. 7.   Effect of phenylephrine and Iso on formation of inositol phosphates. A: [3H]inositol-labeled parotid cells were incubated for 2-20 min in absence of Iso (triangle ) or in presence of 100 nM Iso (black-triangle), 1 µM phenylephrine (open circle ), or Iso + phenylephrine (). B: [3H]inositol-labeled parotid cells were incubated for 10 min in absence of Iso or presence of Iso (100 nM), phenylephrine (1 or 10 µM), or Iso + phenylephrine. Results are expressed as percent increase above basal level (before incubation). Values are means ± SE of 6 independent experiments. IP1, IP2, and IP3, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that the physiological level of beta -adrenergic stimulation potentiates the [Ca2+]i elevation elicited by alpha -adrenergic and muscarinic stimulations in parotid cells. Iso potentiated the [Ca2+]i elevation in a concentration-dependent manner, with half-maximal potentiation at 3 nM. This concentration range of Iso closely agreed with that needed to promote cAMP formation (31) and amylase exocytosis (26). Although it is known that high concentrations of Iso (>10 µM) stimulate alpha -adrenergic receptors, by which [Ca2+]i is increased (28), those concentrations are more than three orders of magnitude higher than that needed to potentiate the Ca2+ response. It is clear from the data here that Iso potentiates the Ca2+ response through the activation of beta -adrenergic receptors. It is also demonstrated that the effect of Iso was mimicked by cAMP-elevating agents, forskolin and DBcAMP, and that it was inhibited by PKA inhibitors, H-89 and K-252a, indicating that the potentiating effect of Iso is mediated by PKA.

In addition, we showed that Iso induces Ca2+ release from intracellular Ca2+ stores through the potentiation of the PLC-dependent signaling pathway, whereas Iso does not alter the Ca2+ response elicited by thapsigargin. These results are strong arguments that the potentiation must be attributed to the enhancement of the Ca2+ release from intracellular Ca2+ stores but not to the enhancement of Ca2+ entry or attenuation of Ca2+ extrusion. Because caffeine failed to induce any Ca2+ response in the absence or presence of Iso, it is verified that ryanodine-sensitive Ca2+ release is not involved in the Ca2+ response in rat parotid cells. Taken together, the results here indicate that PKA potentiates the PLC-dependent Ca2+ release from IP3-sensitive Ca2+ stores in rat parotid cells.

Because phenylephrine-induced production of inositol phosphates was not enhanced by Iso, we speculate that the potentiation of the agonist-induced Ca2+ response can be attributed to the sensitization of the IP3-sensitive Ca2+ release. This would be consistent with an earlier observation by Rubin and Adolf (23) that cAMP potentiates the IP3-induced Ca2+ release in permeabilized parotid cells. Although it has been reported that the PKA-coupled receptor stimulates PLC through beta gamma -subunits of GTP-binding protein (25, 35) or that it sensitizes a PLC-coupled receptor (20, 24), the results here suggest that these types of cross talk are not substantial in rat parotid cells. Because Iso (or cAMP-elevating agents) alone did not change the basal [Ca2+]i, the possibility that the enhanced Ca2+ release is secondary to an inhibition of the Ca2+ uptake can be eliminated. Furthermore, it is also unlikely that the enhanced Ca2+ response can be attributed to an increase in Ca2+ loading and a resultant increase in Ca2+ contents in the stores. Although the increased Ca2+ loading may enhance the peak [Ca2+]i elevation, it should then reduce the sustained [Ca2+]i by sequestration of the released Ca2+. Taken together, our results argue strongly in favor of a direct effect of PKA on the IP3-sensitive Ca2+ channel (IP3 receptor). This type of synergistic cross talk has been reported in hepatocytes (5, 12), pancreatic beta -cells (18), and articular chondrocytes (6). In support of this view, it is known that IP3 receptor protein is a good substrate for PKA (8, 9, 19). In addition, Wojcikiewicz and Luo (33) recently demonstrated that the PKA-dependent phosphorylation of IP3 receptors correlates with the sensitivity for Ca2+ release in several cultured cell types. Although the substrate for PKA causing the potentiation has not been determined, PKA-dependent phosphorylation of IP3 receptors is the most likely mechanism for the synergism between cAMP- and PLC-coupled signaling pathways on the Ca2+ response in rat parotid cells.

The synergistic interaction between cAMP- and PLC-coupled receptors has been reported for many cell types. It has been considered that this synergistic interaction plays a role in the modulation or fine tuning of multiple receptor-signaling pathways (25). It may be postulated that the potentiating effect of PKA for the Ca2+ response is physiologically relevant for the regulation of salivary fluid secretion. In particular, norepinephrine, the neurotransmitter released from sympathetic nerves, stimulates alpha - and beta -adrenergic receptors simultaneously. In fact, Jirakulsomchok and Schneyer (16) reported that the administration of a beta -adrenergic blocker, propranolol, significantly attenuates the salivary fluid secretion elicited by electrical stimulation of sympathetic nerves in rats. In addition, it has been reported that the activation of the cAMP pathway with VIP or forskolin enhances salivary fluid secretion elicited by PLC-coupled receptor agonists, such as substance P, phenylephrine, and carbachol (4, 7, 17). Because Ca2+ is the principal regulator for the salivary fluid secretion, the synergism between the cAMP and PLC pathways on salivary secretion may be thought to be mediated by the potentiation of the PLC-dependent Ca2+ response by PKA.

In conclusion, the present study demonstrated that the beta -adrenergic agonist potentiates the Ca2+ response elicited by PLC-coupled receptor agonists. This potentiation is considered to be mediated by PKA and can probably be attributed to the sensitization of IP3 receptors. This PKA-induced potentiation of the Ca2+ response is thought to account for the synergism between cAMP- and PLC-coupled receptors for salivary fluid secretion.


    FOOTNOTES

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 for reprint requests and other correspondence: A. Tanimura, Dept. of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan (E-mail: tanimura{at}hoku-iryo-u.ac.jp).

Received 9 November 1998; accepted in final form 16 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 276(6):C1282-C1287
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