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Am J Physiol Cell Physiol 277: C262-C270, 1999;
0363-6143/99 $5.00
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Vol. 277, Issue 2, C262-C270, August 1999

Nitric oxide acts independently of cGMP to modulate capacitative Ca2+ entry in mouse parotid acini

Eileen L. Watson1,2, Kerry L. Jacobson1, Jean C. Singh1, and Sabrina M. Ott1

Departments of 1 Oral Biology and 2 Pharmacology, University of Washington, Seattle, Washington 98195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Carbachol- and thapsigargin-induced changes in cGMP accumulation were highly dependent on extracellular Ca2+ in mouse parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) resulted in complete inhibition of agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca2+ release and capacitative Ca2+ entry, whereas the inhibition of sGC had no effect. The effects of NOS inhibition were not reversed by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by the inhibition of sGC. GEA-3162-induced increases in Ca2+ release from ryanodine-sensitive stores and enhanced capacitative Ca2+ entry, both of which were unaffected by inhibitors of sGC but reduced by NOS inhibitors. Results support a role for NO, independent of cGMP, in agonist-mediated Ca2+ release and Ca2+ entry. Data suggest that agonist-induced Ca2+ influx activates a Ca2+-dependent NOS, leading to the production of NO and the release of Ca2+ from ryanodine-sensitive stores, providing a feedback loop by which store-depleted Ca2+ channels are activated.

carbachol; thapsigargin; GEA-3162; nitric oxide synthase inhibitors; calcium ion release


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTRACELLULAR CALCIUM PLAYS a fundamental role in linking receptor stimulation to enzyme secretion in exocrine cells. In nonexcitable cells, the rapid rise in intracellular Ca2+ concentration ([Ca2+]i) is due to the release of Ca2+ from intracellular stores. This is followed by an influx of Ca2+ from the extracellular medium, which lasts for minutes. The depletion of intracellular Ca2+ pools appears to be sufficient to induce capacitative Ca2+ entry (33). An important question relating to Ca2+ signaling has been how the depletion of intracellular Ca2+ stores leads to increased Ca2+ entry. Until recently, the proposed model has been that capacitative Ca2+ entry is due to the generation of an intracellular mediator(s). Candidates include tyrosine kinase or phosphatase (6), Ca2+ influx factor (34), and a GTP-binding protein (4, 12, 47). In addition, Ca2+ entry has been proposed to be activated by cGMP in several cell types including pancreatic acinar cells (2, 17, 30, 31, 48, 49), submandibular cells (49), colonic epithelial cells (5), pituitary GH3 cells (43), and NIE-115 neuroblastoma cells (19), but not in other cell types (3, 7). Further, debate as to the role of cGMP in capacitative Ca2+ entry in the same cell type, e.g., in pancreatic acinar cells (15), still remains. In cells showing a positive correlation between cGMP and Ca2+ entry, the effects of cGMP appear to occur via a signaling pathway involving NO (5, 10, 17, 19). The NO produced after activation of a Ca2+-dependent NOS increases cGMP via activation of a soluble guanylate cyclase (22). In addition to the mediator hypothesis, an alternative model, in which D-myo-inositol 1,4,5-trisphosphate (IP3) receptors in Ca2+ stores are coupled to store-operated channels and Ca2+ release-activated Ca2+ current, has been proposed (24).

NO is an important messenger with complex cellular effects. From a recent review by Clementi (9), it is clear that NO has profound effects on Ca2+ homeostasis. NO is involved in the regulation of voltage-dependent Ca2+ channels (29) and voltage-independent, store-operated Ca2+ channels (2, 48), modulation of IP3-induced intracellular Ca2+ release (25), Ca2+ release from ryanodine stores (32, 39, 44), regulation of Ca2+ influx (2, 5, 10, 17, 19, 28, 48, 49), and IP3 and cyclic ADP-ribose generation (45). Many of these actions appear to be mediated via cGMP through activation of a G-kinase (9) or phosphodiesterase (29). Recently, NO has been shown to produce effects that are independent of cGMP, e.g., direct activation of ryanodine receptors (RyRs) via nitrosylation of regulatory thiols (37, 46).

The goal of the present study was to determine the role of NO in capacitative Ca2+ entry and the underlying molecular mechanism(s) involved. To test the relationship between NO and capacitative Ca2+ entry, we examined the effects of carbachol and the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin on Ca2+ entry in the absence and presence of the nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine (L-NNA) and 7-nitroindazole (7-NI) (1). To determine the mechanism of NO, we examined 1) the effects of the soluble guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione (LY-83583) (35) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (14) as well as the NOS inhibitor 7-NI on agonist- and NO donor-induced Ca2+ release and capacitative Ca2+ entry and 2) the effects of NO on Ca2+ release via ryanodine-sensitive Ca2+ stores and on [3H]ryanodine binding to isolated microsomes. Results suggest that NO, acting independently of cGMP, is involved in capacitative Ca2+ entry by releasing Ca2+ from ryanodine-sensitive Ca2+ stores in mouse parotid cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Materials were obtained as follows: hyaluronidase, carbachol, 8-bromo-cGMP, BSA, IBMX, dithiothreitol (DTT), and HEPES were from Sigma (St. Louis, MO); thapsigargin was from Calbiochem (La Jolla, CA); collagenase type CLS2 was from Worthington (Freehold, NJ); cGMP RIA kits were from New England Nuclear (Boston, MA); fura 2-AM was from Molecular Probes (Eugene, OR); ODQ was from Tocris (Ballwin, MO); LY-83583 was from Biomol Research Laboratories (Plymouth Meeting, PA); L-NNA and 7-NI were from RBI (Natick, MA); and 1,2,3,4-oxatriazolium 5-amino-3-(3,4-dichlorophenyl)-chloride (GEA-3162) was from Alexis (San Diego, CA). All other reagents were of analytical grade or higher.

Preparation of parotid acini. Small groups of isolated mouse parotid cells (acini) were prepared as described previously by Watson et al. (41). Briefly, parotid glands from male Swiss-Webster mice (27-30 g) were removed quickly, trimmed, and minced in a siliconized dish in Krebs-Henseleit bicarbonate solution (KHB), pH 7.4, containing 0.9 mM Mg2+ and 1.28 mM Ca2+, 30 mM HEPES, 90 U/ml collagenase (CLS2), and 1 mg/ml hyaluronidase. Enzyme digestion was conducted in a rotary water bath at 37°C for 60 min under continuous 5% CO2-95% O2 gassing. After the first 40 min of digestion, the suspension was pipetted up and down 12 times with a 10-ml plastic pipette. This was repeated two more times at ~5-min intervals. The pH during the dispersion was maintained at 7.2 to 7.4. After digestion, the cells were centrifuged at 50 g for 2 min, washed with buffer (KHB minus enzymes with 4% BSA, pH 7.4), filtered through two layers of nylon, and washed two additional times. Cells were suspended in KHB minus enzyme containing 1% BSA and rested for 30 min at 37°C with continuous gassing.

Measurement of [Ca2+]i in intact cells. Acini were suspended 1:50 (wt/vol) in KHB containing 0.176 mg/ml ascorbic acid and 0.2% BSA, pH 7.4, and loaded with fura 2-AM at 3.3 µg/ml of cell suspension for 30 min at 37°C with continuous gassing (5% CO2-95% O2) and shaking. Fura 2-AM was prepared at 1 mg/ml in DMSO just before use. Loaded cells were washed three times in the 0.2% BSA-KHB containing ascorbic acid, resuspended at 1:50 (wt/vol), and maintained at 24°C with gassing and shaking. After a 20-min incubation period, an aliquot was washed twice in the above buffer with or without Ca2+, diluted 1:10 in fresh buffer, and placed in ultraviolet-grade fluorometric cuvettes (Spectrocel) for [Ca2+]i measurements. [Ca2+]i was calculated from the equation of Grynkiewicz et al. (16), where the dissociation constant (Kd) = 224 mM. A Filterscan spectrofluorometer system equipped with a magnetic stirrer and constant-temperature cuvette holder from Photon Technology International (South Brunswick, NJ) was used for the [Ca2+]i measurements.

Cyclic nucleotide measurements. cGMP levels in intact mouse parotid acini suspended at ~1:300 (wt/vol) in KHB, pH 7.4, containing 0.1% BSA were measured as described previously (41). Incubations were terminated by the addition of an equal volume of ice-cold 10% TCA. cGMP levels were determined by the RIA procedure of Steiner et al. (36). Samples were acetylated for cGMP determinations as described by Harper and Brooker (18). Results were calculated as femtomoles of cGMP per milligram of protein.

Microsomal membrane preparation. Microsomal membranes were isolated at 4°C from parotid acinar cells by fractionation of a 10% (wet wt/vol) homogenate of the cells by using differential centrifugation in isomolar sucrose as described previously (11). Acini were suspended in a solution containing 250 mM sucrose, 10 mM HEPES, 2 mM EDTA buffer (pH 7.4), and the following protease inhibitors: leupeptin (1 µg/ml), pepstatin A (0.7 µg/ml), and phenylmethylsulfonyl fluoride (PMSF; 0.1 mM) and homogenized by 10 complete passes in a glass mortar with a motor-driven Teflon pestle. The homogenate was centrifuged for 5 min at 250 g. Homogenization of the pellet was repeated, and the pooled 250 g supernatants were centrifuged for 20 min at 10,000 g. The 10,000 g supernatant was centrifuged for 1 h at 100,000 g, and the resulting microsomal fraction (pellet) was separated from the soluble fraction (supernatant), held overnight submerged in suspension buffer (588 mM sucrose, 50 mM KCl, 20 mM MOPS buffer, pH 6.8, containing 1 µg/ml leupeptin and 0.7 µg/ml pepstatin A) at 4°C, and resuspended the next day at a protein concentration of 5-11 mg/ml by gentle homogenization in the same buffer for immediate use or storage at -80°C.

[3H]ryanodine binding assay. [3H]ryanodine binding to mouse parotid acinar cell membranes was performed as described previously (11) except for the binding temperature and duration. Briefly, membrane samples were incubated in binding buffer consisting of 0.5 M KCl, 100 µM CaCl2, 20 mM HEPES (pH 7.4), and protease inhibitors aprotinin (0.5 mg/ml), leupeptin (1 µg/ml), and pepstatin A (0.7 µg/ml) with the protease substrate BSA (100 µg/ml), with or without GEA-3162 for 2 h at 37°C. The IC50 value for GEA-3162 was obtained from concentration-response experiments assessing the binding of [3H]ryanodine added at a nonsaturating concentration of 6 nM. The assay was terminated by rapid dilution of the sample with 4 ml of wash buffer containing 0.5 M KCl, 20 mM HEPES (pH 7.4), and 100 µM Ca2+ and passage of the sample through a Whatman GF/F glass fiber filter, followed immediately by three 4-ml washes of the filter with the same buffer; all procedures were completed within 1 min. The filters were dried overnight and placed in vials containing scintillant, and the bound [3H]ryanodine was measured by liquid scintillation counting with a Packard Tri-Carb 2200CA analyzer. Specific bound [3H]ryanodine was calculated by subtracting nonspecific binding, measured in the presence of 10 µM unlabeled ryanodine.

Protein was determined by the Hartree (20)-modified method of Lowry et al. (26).

Data analysis. cGMP data and [Ca2+]i determinations involving NOS inhibitors and GEA-3162 are presented as means ± SE. Statistical analysis was performed by using Student's t-test (P < 0.05).

The IC50 for GEA-3162 inhibition of [3H]ryanodine binding was determined by linear analysis of the log-logit transformation of concentration-response curves. Binding constants Kd and maximum binding capacity (Bmax) values for [3H]ryanodine with and without GEA-3162 were derived by curvilinear analysis using the computer program RADLIG, subroutine EBDA (Elsevier-BIOSOFT), and were depicted graphically by a Rosenthal (Scatchard) plot and linear analysis. Observed differences in RyR concentration and affinity constants in parotid membranes treated or not treated with GEA-3162 were tested for significance (P < 0.05) by using the computer program LIGAND and F statistics. Values reported represent the means ± SE of experiments performed in duplicate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of Ca2+ release and influx in muscarine- and thapsigargin-induced increases in cGMP accumulation. Previous studies have shown that activation of muscarinic receptors leads to increases in cGMP levels in mouse parotid acini (40, 41). However, the contribution of Ca2+ influx and release from intracellular stores to cGMP accumulation in these cells was not examined. As shown in Fig. 1A, carbachol (10 µM) increased cGMP accumulation significantly in the presence of 1.28 mM extracellular Ca2+ (trace a). cGMP levels increased from 1,178 ± 225 to 3,794 ± 290 fmol/mg protein within 0.75 min and declined slightly by 5 min. In a nominally Ca2+-free medium, however, carbachol-induced cGMP accumulation represented only 12% of the cGMP produced in a Ca2+-replete buffer; cGMP increased from 695 ± 55 to 1,016 ± 74 fmol/mg protein within 0.25 min, then declined rapidly to baseline (trace b). The percent increase in cGMP accumulation in the absence of extracellular Ca2+ was only slightly increased by a higher concentration of carbachol (1 mM), i.e., from 12 to 15%; IBMX (100 µM) had little effect on carbachol (10 µM)-induced cGMP levels, i.e., maximum cGMP levels were increased by 6% (data not shown).


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  		Fig. 1.   Time course of carbachol (10 µM)-induced changes in cGMP accumulation (A) and intracellular Ca2+ concentration ([Ca2+]i) (B) in mouse parotid acini incubated in a 1.28 mM Ca2+-containing buffer (trace a) or a nominally Ca2+-free Krebs-Henseleit bicarbonate (KHB) buffer (trace b). Results are representative of 4 experiments.

To further assess the role of Ca2+ in cGMP accumulation, we used the Ca2+-ATPase inhibitor thapsigargin, which, unlike carbachol, acts independently of the phospholipase C pathway. Like carbachol, thapsigargin increased cGMP levels significantly in the presence of 1.28 mM extracellular Ca2+ (Fig. 2A); cGMP levels increased by 418% from 1,197 ± 99 to 6,201 ± 434 fmol/mg protein in the presence of 1.28 mM extracellular Ca2+ (trace a). In a nominally Ca2+-free medium, cGMP accumulation increased by 73.2% from 741 ± 54 to 1,284 ± 21 fmol/mg protein and represented 11.8% of the cGMP generated in a Ca2+-replete buffer (trace b). Thus, like carbachol, Ca2+ influx was a major contributor to cGMP accumulation. The inclusion of IBMX (100 µM) in the medium had little effect on cGMP levels (data not shown). Unlike what was found for carbachol, maximum cGMP accumulation, induced by thapsigargin in the presence of 1.28 mM Ca2+, was reached at 3 min.


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  		Fig. 2.   Time course of thapsigargin (2 µM)-induced changes in cGMP accumulation (A) and [Ca2+]i (B) in mouse parotid acini incubated in a 1.28 mM Ca2+-containing buffer (trace a) or a nominally Ca2+-free KHB buffer (trace b). Results are representative of 4 experiments.

Effects of inhibitors of the NO/cGMP pathway on stimulated cGMP levels and Ca2+. As shown in Table 1, NOS inhibitors L-NNA (2 mM) and 7-NI (200 µM) and soluble guanylate cyclase inhibitors LY-83583 (30 µM) and ODQ (3-10 µM) completely inhibited carbachol- and thapsigargin-stimulated cGMP production. In parallel studies, using the Ca2+-free/Ca2+ reintroduction protocol described previously (42), these inhibitors were employed to determine the role of NO and cGMP in agonist-induced Ca2+ release and capacitative Ca2+ entry. For the NOS inhibitor studies, L-NNA (2 mM) and 7-NI (200 µM) were preincubated with acini for 10 and 2 min, respectively, before the addition of agonists. In a nominally Ca2+-free buffer, L-NNA (2 mM) reduced carbachol- and thapsigargin-induced Ca2+ release by 31.0 ± 6.4 and 36.0% (n = 2), respectively (Fig. 3, A and B, trace b). After the reintroduction of 1.28 mM Ca2+, capacitative Ca2+ entry was reduced by 39.5 ± 4.8 and 31.7 ± 3.5%, respectively. Trace c represents the control. Similarly, 7-NI (200 µM) reduced carbachol- and thapsigargin-induced Ca2+ release by 42.8 ± 5.7 and 44.3 ± 6.7%, respectively (P < 0.05), and capacitative Ca2+ entry by 44.2 ± 3.0 and 54.3 ± 4.3%, respectively (P < 0.05) (Fig. 4, A and B, trace b). Trace c represents the control.

                              
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  		Table 1.   Effects of NOS and soluble guanylate cyclase inhibitors on cGMP accumulation



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  		Fig. 3.   Effects of nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) on carbachol- (A) and thapsigargin (B)-induced Ca2+ release and capacitative Ca2+ entry in mouse parotid acini. Trace a: carbachol (10 µM) or thapsigargin (2 µM) was added at 180 s to acini incubated in a nominally Ca2+-free KHB buffer; this was followed by reintroduction of 1.28 mM Ca2+ at 350 s (A) or 420 s (B). Trace b: L-NNA (2 mM) was added to acini for 10 min before addition of agonist. Trace c (control): acini were incubated with L-NNA (2 mM) alone; this was followed by reintroduction of 1.28 mM Ca2+. Results are representative of 4 experiments.



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  		Fig. 4.   Effect of NOS inhibitor 7-nitroindazole (7-NI) on carbachol- (A) and thapsigargin (B)-induced Ca2+ release and capacitative Ca2+ entry in mouse parotid acini. Trace a: carbachol (10 µM) or thapsigargin (2 µM) was added at 180 s to acini incubated in a nominally Ca2+-free KHB buffer; this was followed by reintroduction of 1.28 mM Ca2+ at 360 s (A) or 420 s (B). Trace b: 7-NI (200 µM) was added to acini 2 min before addition of agonist. Trace c (control): acini were incubated with 7-NI (200 µM) alone; this was followed by reintroduction of 1.28 mM Ca2+. Results are representative of 4 experiments.

To determine whether cGMP could reverse the effects of NOS inhibition, we examined the effect of 8-bromo-cGMP (0.5 mM) on thapsigargin-induced Ca2+ release and Ca2+ entry in the presence of 7-NI (200 µM). As shown in Fig. 5A, 8-bromo-cGMP added 10 min before the addition of thapsigargin (2 µM) did not reverse 7-NI inhibition of either thapsigargin-induced Ca2+ release or capacitative Ca2+ entry (trace c). ODQ (3 µM), an inhibitor of NO-stimulated guanylate cyclase (14), had no effect on thapsigargin-induced responses (Fig. 5B, trace b). There was little increase in Ca2+ entry in control acini (data not shown; refer to Figs. 4, A and B, and 8). LY-83583 was not used in these studies because it reduced GEA-3162-induced cGMP accumulation by only 37% (Table 1). The inability of LY-83583 to completely inhibit GEA-3162-induced cGMP accumulation may be because part of its action is to block NOS activity (27) as well as that of soluble guanylate cyclase; blocking NOS activity would not interfere with the actions of NO donors.


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  		Fig. 5.   Effects of 8-bromo-cGMP on 7-NI inhibition of thapsigargin-induced Ca2+ release and capacitative Ca2+ entry (A), and effects of ODQ on thapsigargin-induced responses (B) in mouse parotid acini. A, trace a: thapsigargin (2 µM) was added at 180 s to acini incubated in a nominally Ca2+-free KHB buffer; this was followed by reintroduction of 1.28 mM Ca2+ at 400 s. Trace b: 7-NI (200 µM) was added 2 min before addition of thapsigargin. Trace c: 8-bromo-cGMP (500 µM) was added 8 min before 7-NI. B, trace a: thapsigargin (2 µM) was added at 180 s; this was followed by reintroduction of 1.28 mM Ca2+ at 420 s. Trace b: ODQ (3 µM) was added 10 min before addition of thapsigargin. Results are representative of 3 experiments.

Effects of NO donors on cGMP levels and [Ca2+]i. To further determine the role of the NO/cGMP pathway in capacitative Ca2+ entry, we first examined the ability of exogenous NO, in the form of the NO-releasing compound GEA-3162, to increase cGMP levels. GEA-3162 has been characterized by its ability to generate nitrate and nitrite in aqueous solution in a time-dependent manner and to increase cGMP levels (23). The production of nitrites and nitrates was much slower with GEA-3162 than with the NO donor S-nitroso-N-acetylpenicillamine, which may be due to fast decomposition of GEA-3162 in aqueous solution (23). An advantage of using GEA-3162 is that, in contrast to other NO donors, such as 3-morpholinosydnonimine, it produces negligible amounts of peroxynitrite (ONOO-) (21), which has cellular effects that previously may have been attributed to NO.

cGMP accumulation in the presence of GEA-3162 (100 µM) was rapidly and significantly increased (Fig. 6A), as was shown previously by Kankaanranta et al. (23); at 0.5 min, cGMP levels increased from 877 ± 386 to 31,246 ± 410 fmol/mg protein. These effects were independent of extracellular [Ca2+]i (data not shown). By 5 min, cGMP levels were reduced to 11,433 ± 376 fmol/mg protein. ODQ (10 µM) reduced GEA-3162-induced cGMP accumulation by ~90% (Table 1), whereas 7-NI and L-NNA were without effect (Table 1).


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  		Fig. 6.   Time course of GEA-3162 on cGMP accumulation (A) and [Ca2+]i (B) in mouse parotid acini. For cGMP determinations GEA-3162 (100 µM) was added at time 0 to acini incubated in a 1.28 mM Ca2+-containing KHB buffer. For the [Ca2+]i determinations GEA-3162 (100 µM) was added to acini incubated in a 1.28 mM Ca2+-containing KHB buffer (trace a) or a nominally Ca2+-free KHB buffer (trace b). Trace c (control): acini incubated without GEA-3162. Results are representative of 5 experiments.

In other studies, GEA-3162 was used to assess the effects of NO on [Ca2+]i. In contrast to the rapid effects of GEA-3162 on cGMP accumulation, GEA-3162 (100 µM) produced a slow but significant increase in [Ca2+]i in a 1.28 mM Ca2+-containing KHB buffer; [Ca2+]i increased from 80 to 250 nM over 15 min (Fig. 6B, trace a), and the increase was similar to the maximal increases produced by carbachol and thapsigargin (see Figs. 1B and 2B). A slow increase in [Ca2+]i by GEA-3162 has been previously reported by Favre et al. (13) for DDT1MF-2 cells and may be related to the fact that GEA-3162, although lipophilic, releases nitrates and nitrites slowly in aqueous solutions (23). The differences noted in the time required for production of cGMP accumulation and increases of [Ca2+]i suggest that the release of low levels of nitrites and nitrates by GEA-3162 is sufficient to rapidly induce cGMP accumulation, whereas greater amounts of nitrites and nitrates are required for observing significant changes in [Ca2+]i. Further, data are also consistent with the premise that the effects of GEA-3162 on [Ca2+]i are independent of cGMP.

To determine whether the NO-induced Ca2+ response was due to Ca2+ release from intracellular stores or to Ca2+ influx, experiments were conducted with a nominally Ca2+-free buffer. The response to GEA-3162 in a nominally Ca2+-free buffer was ~40% of that found in the presence of 1.28 mM Ca2+; the average of four experiments was 45.6 ± 6.0% (P < 0.05). As shown in Fig. 6B (trace b), [Ca2+]i increased from 50 to 150 nM. Because 7-NI was found to reduce carbachol- and thapsigargin-induced Ca2+ release, in addition to capacitative Ca2+ entry, the data suggested that NOS may be involved in the agonist-induced release of Ca2+ from intracellular stores. Therefore, because GEA-3162 released Ca2+ from intracellular stores, we determined whether 7-NI would also inhibit this response. 7-NI (200 µM) added 2 min before GEA-3162, significantly reduced the effects of GEA-3162 on Ca2+ release (P < 0.05; Fig. 7, trace b). The average of four experiments was 77.2 ± 5.4% compared with 42.8 ± 5.7 and 44.3 ± 6.7% for carbachol and thapsigargin, respectively (Fig. 4, A and B, trace b), confirming an involvement of NOS in this process.


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  		Fig. 7.   Effects of 7-NI on GEA-3162-induced Ca2+ release. Trace a: GEA-3162 (100 µM) was added at 60 s to acini incubated in a nominally Ca2+-free KHB buffer. Trace b: 7-NI (200 µM) was added 2 min before addition of GEA-3162. Trace c (control): acini incubated with 7-NI (200 µM) alone. Results are representative of 3 experiments.

To assess whether GEA-3162-induced Ca2+ release activates capacitative Ca2+ entry, acini were treated with GEA-3162 before the reintroduction of 1.28 mM Ca2+. As shown in Fig. 8, GEA-3162 produced a gradual increase in [Ca2+]i in a nominally Ca2+-free KHB buffer (trace a). The reintroduction of 1.28 mM Ca2+ caused a rapid, significant increase in [Ca2+]i that was followed by a slower, sustained rise in [Ca2+]i (trace a); there was little increase in Ca2+ entry in control acini (trace c). ODQ (3 µM) had no effects on GEA-3162-induced Ca2+ release or capacitative Ca2+ entry (trace b), supporting the premise that NO acts independently of cGMP to produce these effects.


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  		Fig. 8.   Effects of GEA-3162 on Ca2+ release and capacitative Ca2+ entry in mouse parotid acini in absence and presence of ODQ. Baseline was recorded to 60 s. At this time, DMSO (trace a) or ODQ (3 µM) (trace b) was added for 10 min (but not recorded), and then GEA-3162 (100 µM) was added to acini incubated in a nominally Ca2+-free KHB buffer for a total of 15 min (5 min of which were not recorded). Ca2+ (1.28 mM) was reintroduced at 660 s (trace a). Trace c (control): acini incubated with ODQ alone. Results are representative of 3 experiments.

Effects of NO on RyRs. Because RyRs have been characterized in mouse parotid cells (11) and NO has been reported to release Ca2+ from ryanodine-sensitive stores (10, 32), we examined the effects of GEA-3162 on RyRs by determining 1) the effects of ryanodine on GEA-3162-induced Ca2+ release and capacitative Ca2+ entry and 2) the binding of radiolabeled [3H]ryanodine to microsomal vesicles. As shown in Fig. 9A (trace b), incubation of acini for 1 h with 200 µM ryanodine reduced GEA-3162 (100 µM)-induced Ca2+ release, which was more easily seen in the reduction in capacitative Ca2+ entry (~27%). This low degree of inhibition may be related to the difficulty of ryanodine to penetrate acini or the difficulty in achieving conditions that are used to favor binding in cell-free systems. Because of the low level of inhibition in intact cells, further studies were conducted in vitro to directly determine whether NO interacts with the RyR. As shown in Fig. 9B, in a cell-free system, GEA-3162 inhibited, in a concentration-dependent manner, [3H]ryanodine binding to microsomes incubated for 2 h at 37°C; the IC50 for two experiments was 20.7 µM (values were 20.5 and 20.9 µM). The incubation of microsomes at earlier time periods produced similar results; however, the inhibition was less pronounced (data not shown). At a concentration shown to decrease occupancy by 50%, GEA-3162 had no significant effect (P > 0.05) on Kd; values were 7.0 ± 0.5 and 9.4 ± 1.2 nM in the absence and presence of GEA-3162, respectively. However, Bmax was reduced significantly (P < 0.01) by 34% (Fig. 10). Values were 332 ± 11 and 231 ± 24 fmol/mg protein in the absence and presence of GEA-3162, respectively. As shown in Fig. 9B (inset), the inhibition of [3H]ryanodine binding by GEA-3162 was significantly diminished in the presence of the sulfhydryl-reducing agent DTT (1 mM).



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  		Fig. 9.   Effect of ryanodine on GEA-3162-induced Ca2+ release and capacitative Ca2+ entry in mouse parotid acini (A) and effects of increasing concentrations of GEA-3162 on [3H]ryanodine binding to acinar cell microsomal membranes (B). A: acini were incubated in nominally Ca2+-free KHB buffer for 1 h before addition of GEA-3162 (100 µM). Baseline (after 1-h incubation) was recorded to 60 s; GEA-3162 (100 µM) was added for a total of 15 min (5 min not recorded); Ca2+ was reintroduced at 660 s (trace a). Ryanodine (200 µM) was present throughout the 1-h incubation (trace b). Trace c (control): acini incubated with ryanodine alone. Data are representative of 3 experiments. B: mouse parotid microsomal membranes (100 µg protein) were incubated for 2 h with 0-3,000 µM GEA-3162 at 37°C. Inset: acini were incubated in absence or presence of dithiothreitol (DTT; 1 mM) for 2 min before addition of GEA-3162. See MATERIALS AND METHODS for further details. Data represent 2 experiments performed in duplicate.



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  		Fig. 10.   Rosenthal (Scatchard) plot of equilibrium binding of 0.3-30 nM [3H]ryanodine to acinar cell microsomal membranes in absence and presence of GEA-3162 (20.7 µM). See MATERIALS AND METHODS for further details. Values are means ± SE from 4 experiments performed in duplicate. Kd, dissociation constant; Bmax, maximum binding capacity; r, correlation coefficient.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The link that communicates the filling state of intracellular Ca2+ stores to the plasma membrane has been the focus of a number of recent studies. Although there is sufficient data to implicate NO as a key player in the generation of cGMP in exocrine cells, as well as other cell types (17, 38, 48), and to support a role for cGMP in capacitative Ca2+ entry (2, 5, 17, 19, 30, 31, 38, 48, 49), these studies have been challenged by negative findings from other laboratories (3, 7, 15). If capacitative Ca2+ entry is mediated by cGMP, it would be expected that Ca2+ released from intracellular stores would increase cGMP levels, cGMP analogues would mimic the effects of agonists on Ca2+ entry, and agonist-mediated Ca2+ entry would be inhibited by agents that block cGMP accumulation. In pancreatic acinar cells, Gilon et al. (15) showed that cGMP was not produced in the absence of extracellular Ca2+ and concluded that cGMP could not be a mediator of Ca2+ entry. In contrast, other data from pancreatic acinar cells support the view that Ca2+ released from intracellular stores is sufficient to activate NOS, leading to increases in cGMP levels (31, 48).

Data from mouse parotid acini clearly indicate that capacitative Ca2+ entry plays the major role in the activation of NOS leading to cGMP accumulation. The location of NOS, i.e., neuronal NOS (nNOS), identified in another exocrine cell, i.e., the submandibular cell (49), close to the plasma membrane where Ca2+ channels reside supports the greater role of Ca2+ influx in cGMP accumulation. Our data also suggest that Ca2+ released from intracellular stores may contribute, at least partially, to the increase in agonist-induced cGMP levels and, under similar conditions used by Gilon et al. (15), in pancreatic acini. However, we did not find any evidence to support a role for cGMP in capacitative Ca2+ entry; cGMP analogues failed to increase [Ca2+]i or reverse the effects of NOS inhibitors on Ca2+ entry. We did find, however, that NOS inhibitors L-NNA and 7-NI blocked, to some extent, agonist-induced Ca2+ entry in mouse parotid acini, as they were reported to have blocked entry in pancreatic acini (15). These inhibitors have been widely used to study the role of cGMP in various cellular processes including capacitative Ca2+ entry. L-NNA is less selective in that it inhibits more than one NOS isoform. 7-NI was used because of its specificity for nNOS, which has been reported to be present in plasma membranes of submandibular acinar cells (46). Because 7-NI is a selective inhibitor of nNOS, data would suggest that nNOS plays an important role in capacitative Ca2+ entry in mouse parotid acini. LY-83583 was also able to partially block thapsigargin-induced capacitative Ca2+ entry in Jurkat T lymphocytes (3), which did not respond to cGMP. The fact that LY-83583 has been found to inhibit nNOS (27), an isoform shown to be present in high levels in secretory cells (49), suggests that NO rather than cGMP is involved in capacitative Ca2+ entry in these cell types. Similar effects of NO on capacitative Ca2+ entry have also been reported for endothelial cells (39). This conclusion is further supported by data from mouse parotid acini showing that Ca2+ entry, induced in the presence of the NO donor GEA-3162, is not inhibited by the specific guanylate cyclase inhibitor ODQ. ODQ has been reported to have no effects on particulate guanylate or adenylyl cyclases, it does not interfere with the steps leading to NO synthesis, it does not block actions of NO that are unrelated to guanylate cyclase activation, and it is the first inhibitor to act on the NO receptor soluble guanylate cyclase (14).

Although it is difficult to explain the differences in the effects of NO and cGMP on capacitative Ca2+ entry in the same cell type, i.e., pancreatic acinar cells, Xu et al. (49) recently suggested that a contributing factor may be the state of the cells. Gilon et al. (15) reported only a 1.2- to 1.4-fold stimulation of cGMP by carbachol and thapsigargin compared with the higher levels of cGMP reported by Xu et al. (48) and Gukovskaya and Pandol (17). This may account for the observed differences in pancreatic cells; however, it is clear that cGMP is not involved in Ca2+ entry in the mouse parotid acini even when the degree of increase in cGMP induced by carbachol and thapsigargin is comparable to that observed in pancreatic cells (49). One possibility, as suggested by Bischof et al. (7), to explain an effect of cGMP on capacitative Ca2+ entry in gastrointestinal cells, but not in HEK-293 and HEK-293/NOS cells, is that some key component is missing in parotid cells that is present in colonic and pancreatic cells. However, it is more likely that the effects of cGMP on capacitative Ca2+ influx may be tissue specific. This would account for differences in the effects of cGMP on Ca2+ entry observed in parotid vs. pancreatic acinar cells, as well as differences between different salivary cells, i.e., parotid and submandibular cells (49). These differences could be explained on the basis that NO has independent effects as well as cGMP-dependent effects.

Of particular importance are questions relating to the mechanism(s) by which NO is involved in capacitative Ca2+ entry. As discussed above, it is clear that cGMP is not involved. Data also do not support a direct role of NO on capacitative Ca2+ entry. The data do suggest, however, that capacitative Ca2+ entry is primarily responsible for activation of NOS and that once activated, the NO produced acts to release Ca2+ from ryanodine stores, setting up a positive feedback loop by which store-operated Ca2+ channels are activated. This conclusion is supported by 1) data showing that in a nominally Ca2+-free KHB buffer, the NO donor GEA-3162 releases significant amounts of Ca2+ from intracellular stores leading to increases in Ca2+ influx when Ca2+ is reintroduced, 2) studies showing that RyRs are present in mouse parotid acini (11) and that ryanodine blocks the effects of NO on Ca2+ release, and 3) [3H]ryanodine-binding studies showing that NO directly interacts with the Ca2+ release protein/ryanodine receptor in mouse parotid acini. Previous studies have shown that nitrosothiol formation underlies the direct modifying effects of NO on a number of channels including the ryanodine channel (37, 46). Further, Favre et al. (13) reported that NO donor GEA-3162-induced Ca2+ entry is activated by S-nitrosylation. Data showing the reversal of GEA-3162-induced inhibition of [3H]ryanodine binding by the sulfhydryl-reducing agent DTT is consistent with S-nitrosylation of the RyR in mouse parotid acini. The finding that GEA-3162 alters Bmax without a change in Kd is consistent with studies by Stoyanovsky et al. (37), who used NO-related compounds to activate skeletal RyRs. On the basis of these studies, as well as our studies, it is suggested that NO produced by GEA-3162 directly interacts with a site on the Ca2+ release protein/RyR (which activates the channel) and prevents the binding of ryanodine to this site.

Release of Ca2+ from intracellular stores by NO is consistent with similar findings reported for other cell types including endothelial cells (39), rat pancreatic beta -cells (44), and interstitial cells from canine colon (32), and with the ability of ryanodine to block NO-induced Ca2+ release (Fig. 9A, trace b) (32, 44). It is unlikely that NO releases Ca2+ from IP3-sensitive Ca2+ stores, as NO has been reported to inhibit the IP3 receptor (8).

In addition to NOS activation by capacitative Ca2+ entry, NOS also appears to be activated by Ca2+ released from intracellular stores. The finding that the NOS inhibitor 7-NI reduces agonist- and GEA-3162-induced Ca2+ release in a nominally Ca2+-free buffer is consistent with an involvement of NOS in the release process. Data suggest that NOS activated by intracellularly released Ca2+ produces NO, which causes a further release of stored Ca2+. Thus the direct interaction of NO with RyRs may serve as an important signaling mechanism to modulate rather than to mediate capacitative Ca2+ entry in mouse parotid acini. It is clear that the cellular effects of NO are complex and depend on the cell type and perhaps the levels of NO. As suggested by Clementi (9), NO appears to be part of an on/off switch mechanism devoted to the fine tuning of the opening of store-operated Ca2+ channels.


    ACKNOWLEDGEMENTS

We thank Dennis H. DiJulio for his assistance in data analysis.


    FOOTNOTES

This work was supported by National Institute of Dental Research Grant DE-05249.

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: E. L. Watson, Dept. of Oral Biology, Box 357132, Univ. of Washington, Seattle, WA (E-mail: ewatson{at}u.washington.edu).

Received 23 December 1998; accepted in final form 28 April 1999.


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