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Am J Physiol Cell Physiol 274: C557-C565, 1998;
0363-6143/98 $5.00
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Vol. 274, Issue 3, C557-C565, March 1998

Capacitative Ca2+ entry is involved in cAMP synthesis in mouse parotid acini

Eileen L. Watson1,2, Zhiliang Wu2, Kerry L. Jacobson1, Daniel R. Storm2, Jean C. Singh1, and Sabrina M. Ott1

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Muscarinic receptor interaction leading to augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The effectiveness of capacitative Ca2+ entry and intracellular Ca2+ release on this response was determined in time course studies by using three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic agonist carbachol, thapsigargin, and ionomycin. Time course studies revealed that Ca2+ release from intracellular stores by carbachol produced an early rapid increase (0.25-0.5 min) in stimulated cAMP levels, whereas capacitative Ca2+ entry resulted in a sustained increase in stimulated cAMP levels that was blocked by La3+. Capacitative Ca2+ entry, alone, was involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. The inability of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and milrinone, to prevent agonist augmentation of cAMP levels, as well as the finding that the type VIII adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this adenylyl cyclase isoenzyme.

thapsigargin; carbachol; ionomycin; phosphodiesterase; intracellular calcium ion stores; adenylyl cyclase; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MUSCARINIC RECEPTORS COUPLE to biochemical effector systems, causing stimulation of phosphoinositide (PI) turnover, inhibition of adenylyl cyclase (AC), activation of guanylate cyclase, and regulation of ion channels (19). In addition, these receptors are also linked to activation of phospholipase A2 (13) and phospholipase D (12) and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (28, 30). In the mouse parotid gland, muscarinic receptor interaction has been shown to augment forskolin and isoproterenol-stimulated cAMP accumulation by mechanisms involving Ca2+ (28, 30). Because direct coupling between muscarinic receptors and stimulation of AC was not observed in membranes from these cells, the response has been interpreted to be the result of cross talk between the PI and AC systems. This is supported by data that show that Ca2+, generated on muscarinic receptor interaction, stimulates AC (21, 30) and enhances the effects of forskolin on cyclase activity in isolated membranes (30). Other studies indicate that the interactions between the cAMP and Ca2+ pathways in parotid cells are more complex. In the presence of isoproterenol, muscarinic receptor interaction leads to biphasic effects on cAMP accumulation, i.e., augmentation and inhibition that is dependent on agonist concentration. These responses are linked to the M3 receptor subtype in parotid acinar cells (27). Inhibition of stimulated cAMP accumulation appears to involve the activation of enzymes that degrade cAMP (28).

The goal of the present study was to determine the contribution of capacitative Ca2+ entry and Ca2+ release from intracellular stores in muscarinic augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini. To explore this relationship, three independent tools were used to manipulate free intracellular Ca2+ concentration ([Ca2+]i): the muscarinic agonist carbachol, the microsomal Ca2+-ATPase inhibitor thapsigargin, and the Ca2+ ionophore ionomycin. In parallel time course studies, changes in capacitative Ca2+ entry and Ca2+ release were correlated with changes in cAMP accumulation. Phosphodiesterase (PDE) inhibitors were utilized to determine the involvement of the enzymes that synthesize and degrade cAMP. Time course studies reveal that Ca2+ release from intracellular stores by carbachol is sufficient to augment stimulated cAMP levels at early time periods (0.25-0.5 min), whereas capacitative Ca2+ entry is required for the sustained increase in stimulated cAMP levels. Capacitative Ca2+ entry, alone, is involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. Failure of PDE inhibitors to reverse agonist augmentation of stimulated cAMP accumulation and the finding that ACVIII is expressed in mouse parotid acini suggest that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this AC isoenzyme.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials were obtained as follows: hyaluronidase, carbachol, isoproterenol, La3+, bovine serum albumin (BSA), ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 3-(N-morpholino)propanesulfonic acid (MOPS), and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma Chemical (St. Louis, MO); cAMP radioimmunoassay kits were from Incstar (Stillwater, MN); collagenase type CLS2 was from Worthington (Freehold, NJ); thapsigargin and ionomycin were from Calbiochem (La Jolla, CA); acetoxymethyl ester of fura 2 (fura 2-AM) was from Molecular Probes (Eugene, Oregon); milrinone was from Biomol (Plymouth Meeting, PA); the vertical gel electrophoresis apparatus, PosiBlot pressure blotter, and ultraviolet Stratalinker were from Stratagene (La Jolla, CA); and oligo(dT) cellulose columns were from Pharmacia LKB Biotechnology (Piscataway, NJ). 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. (28) with modification. 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 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 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 7.2-7.4. 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 buffer containing 1% BSA and were rested for 30 min at 37°C with continuous gassing.

Cyclic nucleotide measurements. cAMP levels were measured in intact mouse parotid acini suspended 1:300 (wt/vol) in KHB buffer, pH 7.4, containing 0.1% BSA as described previously (30). For experiments in which La3+ (LaCl3) was used, a phosphate and bicarbonate-free buffer was used. Cell suspensions (1,500 µl) were incubated with agonists for varying times up to 5 min. Incubations were terminated by addition of an equal volume of ice-cold 10% trichloroacetic acid. cAMP was determined by the radioimmunoassay procedure of Steiner et al. (24). Results were calculated as picomoles of cAMP per milligram of protein. Protein was determined by the method of Lowry et al. (17).

Measurement of [Ca2+]i in intact cells. Acini were suspended 1:50 (wt/vol) in KHB buffer containing 0.176 mg/ml ascorbic acid and 0.1% BSA, pH 7.4, and were loaded with fura 2-AM at 3.3 µg/ml cell suspension for 30 min at 37°C with continuous gassing (95% O2-5% CO2) and shaking. Fura 2-AM was prepared at 1 mg/ml in dimethyl sulfoxide just before use. Loaded cells were washed three times in the 0.1% BSA/KHB buffer 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 and without Ca2+ (diluted 1:10) and placed in ultraviolet grade fluorometric cuvettes (Spectrocel) for [Ca2+]i measurements. For experiments in which LaCl3 was used, a phosphate- and bicarbonate-free buffer (3) was prepared. A 2-min temperature equilibration period was observed before commencing the experiment. Cells were used within a 2.5-h period. [Ca2+]i was calculated using the equation of Grynkiewicz et al. (11), where dissociation constant (Kd) = 224 nM. A Filterscan spectrofluorometer system equipped with a magnetic stirrer and constant temperature cuvette holder from Photon Technology International (S. Brunswick, NJ) was used for the [Ca2+]i measurements.

Northern analysis. Total RNA was isolated from frozen parotid tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (7). Poly(A)+-selected RNA was isolated from total RNA using type III oligo(dT) cellulose (Collaborative Research, Bedford, MA) and was analyzed on a formaldehyde-1.2% agarose gel in MOPS buffer (1). mRNA was transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH) in 10× saline sodium citrate (SSC) for 16-20 h, cross-linked at 80°C for 1 h, and prehybridized at 42°C in a hybridization buffer containing 50% formamide, 5× SSC, 1× Denhardt's solution, and 250 µg/ml denatured salmon sperm DNA for 4 h. [alpha -32P]dCTP random-primed cDNA probes generated from the first 771 nucleotides of the coding region and 329 nucleotides of the 5'-untranslated region of the ACVIII were used for Northern analysis. These probes were hybridized to the immobilized RNA for 16-20 h at 42°C in the hybridization buffer. The membrane was washed in 2× SSC/0.5% SDS for 10 min at room temperature and once for 5 min at 65°C. It was then washed three times for 20 min in 1× SSC/0.5% SDS at 65°C before autoradiography. Phosphorimager quantitation was performed by exposing the radioactive blot to a phosphor screen. After appropriate exposure, the screens were scanned using the phosphorimager model 400S (Molecular Dynamics, Sunnyvale, CA).

Data analysis. Data are presented as means ± SE. Statistical analysis was performed using a paired Student's t-test.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Relationship between muscarinic augmentation of isoproterenol-stimulated cAMP accumulation and [Ca2+]i. In previous studies, we found that muscarinic receptor interaction produced a twofold increase in cAMP levels in the presence of the PDE inhibitor IBMX (30). Data also showed that Ca2+ stimulation of cAMP levels is evident without PDE inhibition, provided that the enzyme is stimulated with another effector (30). In the presence of isoproterenol, carbachol produced a biphasic effect on cAMP accumulation, i.e., augmentation at low concentrations of carbachol (0.1-10 µM) and a decrease in cAMP levels at carbachol concentrations >10 µM; the former was reported to involve Ca2+ and the latter was found to be independent of Ca2+ and due to activation of a PDE isoenzyme(s) (28).

To examine the contribution of Ca2+ entry and Ca2+ release from intracellular stores in muscarinic augmentation of stimulated cAMP accumulation, the time course of effects of carbachol (10 µM) on isoproterenol (0.1 µM)-stimulated cAMP levels was examined. The rationale for conducting time course experiments was based on previous studies that were designed to determine the mechanism involved in carbachol inhibition of cAMP accumulation. In these time course studies, two phases of cAMP accumulation were revealed: an early rapid (0.25-0.5 min) increase in cAMP that was subsequently followed by inhibition of stimulated cAMP accumulation (28). As shown in Fig. 1A, under conditions where carbachol augmented stimulated cAMP accumulation, an early rapid increase (0.25-0.5 min) in cAMP levels was also noted. This was followed by a sustained increase in cAMP levels measured over a 5-min period. Removal of extracellular Ca2+ did not decrease the early rapid rise in cAMP levels but resulted in a reduction of the sustained phase of cAMP accumulation to levels produced by isoproterenol alone (Fig. 1B). Pretreatment of acini with 2 µM thapsigargin for 10 min before the addition of carbachol plus isoproterenol significantly reduced the early rapid phase of cAMP accumulation as described previously (28), suggesting that this phase was due to the release of Ca2+ from intracellular stores.


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  		Fig. 1.   Time course of cAMP accumulation in mouse parotid acini stimulated by isoproterenol (Iso, 0.1 µM) and isoproterenol + carbachol (Carb, 10 µM) in a Ca2+ (1.28 mM)-containing Krebs-Henseleit bicarbonate (KHB) buffer (A); and a Ca2+-free KHB buffer containing 0.5 mM EGTA (B). Results are representative of at least 10 experiments.

To assess whether capacitative Ca2+ entry was involved in the sustained phase of cAMP accumulation, we first determined whether capacitative Ca2+ entry was operative in mouse parotid acini. Fura 2-AM-loaded acini were suspended in KHB buffer containing 1.28 mM Ca2+ for 1 min, followed by the addition of carbachol (10 µM). Carbachol produced a rapid increase in [Ca2+]i, from a resting value of 80 to 300 nM. Addition of 1.5 mM EGTA rapidly reduced [Ca2+]i to resting levels; reintroduction of Ca2+ caused a rapid rise in Ca2+ entry that was concentration dependent (Fig. 2). These data are consistent with capacitative entry as proposed by Putney (23). Because it was important to examine the role of both Ca2+ release and capacitative Ca2+ entry in cAMP accumulation, further studies were conducted in a Ca2+-free KHB buffer using the Ca2+-free/Ca2+ reintroduction protocol commonly used to determine capacitative Ca2+ entry (8). Acini were suspended in a Ca2+-free KHB buffer containing 0.5 mM EGTA for 1 min, and, as shown in Fig. 3A, carbachol (10 µM) produced a rapid increase in [Ca2+]i, which returned to basal levels after 3 min. This increase in [Ca2+]i represents the emptying of intracellular Ca2+ pools. Reintroduction of Ca2+ (1.28 mM) to the buffer induced a rapid and sustained rise in [Ca2+]i due to capacitative Ca2+ entry that was blocked by La3+ (50 µM).


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  		Fig. 2.   Effects of carbachol on free intracellular Ca2+ concentration ([Ca2+]i) in mouse parotid acini in a buffer containing Ca2+ (1.28 mM) with 1.5 mM EGTA at 180 s; 3.0 mM (a), 1.28 mM (b), and 0.5 mM (c) Ca2+ were added at 300 s. Results are representative of 4 experiments.


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  		Fig. 3.   Effects of carbachol on [Ca2+]i and stimulated cAMP accumulation in mouse parotid acini in a Ca2+-free KHB buffer. A, top: carbachol (10 µM) was added at 60 s and 1.28 mM Ca2+ was added at 360 s (trace a). In trace b, La3+ (50 µM) was added 2 min before addition of Ca2+. A, bottom (control): acini were incubated with 50 µM La3+ at 240 s, and 1.28 mM Ca2+ was added at 360 s. B: isoproterenol (0.1 µM) + carbachol (10 µM) were added for 1 min in absence of Ca2+, followed by reintroduction of 1.28 mM Ca2+ (trace a), no addition of Ca2+ (trace b), or reintroduction of 1.28 mM Ca2+ in presence of La3+ (50 µM; trace c). Results are representative of 3 experiments.

In parallel cAMP time course experiments (Fig. 3B), the Ca2+-free/Ca2+ reintroduction protocol was also used to evaluate the role of Ca2+ release and capacitative Ca2+ entry in carbachol augmentation of stimulated cAMP accumulation. These experiments allowed examination of both the early rapid and sustained phases of cAMP accumulation during the initial rapid and sustained increases in [Ca2+]i. In a Ca2+-free KHB buffer containing 0.5 mM EGTA, the simultaneous addition of carbachol plus isoproterenol produced an early rapid rise in cAMP (see Fig. 3B). This was followed by a decrease in cAMP levels over 5 min to levels observed for isoproterenol alone (see Fig. 1B). As stated above, this inhibition of cAMP accumulation is due to activation of a PDE isoenzyme (28). Reintroduction of 1.28 mM Ca2+ to the incubation buffer at 1 min, however, reversed the inhibition and caused a significant sustained increase in cAMP accumulation. The sustained increase in cAMP levels produced by carbachol was blocked by La3+ (50 µM), confirming that capacitative Ca2+ entry was required for this phase of cAMP accumulation.

Relationship between thapsigargin augmentation of isoproterenol-stimulated cAMP accumulation and [Ca2+]i. Thapsigargin was considered a useful tool for examining the relationship between Ca2+ entry and release on stimulated cAMP accumulation because it depletes intracellular Ca2+ pools independently of receptor activation and PI production (26). Any potential effects of protein kinase C (PKC) and beta gamma -subunits of G proteins would thus be eliminated. Time course studies, presented in Fig. 4, show that thapsigargin (2 µM) mimicked the effects of carbachol on the sustained phase of cAMP accumulation. The thapsigargin response was slower in onset than that produced by carbachol, and an early rapid increase (0.25-0.5 min) in cAMP was not observed.


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  		Fig. 4.   Time course of cAMP accumulation in mouse parotid acini stimulated by isoproterenol (0.1 µM) and isoproterenol + thapsigargin (Thaps, 2 µM) in a Ca2+ (1.28 mM)-containing KHB buffer. Results are representative of at least 10 experiments.

To further assess whether capacitative Ca2+ entry was involved in thapsigargin augmentation of the sustained phase of cAMP accumulation, the effects of thapsigargin on [Ca2+]i were compared with effects on cAMP accumulation using the Ca2+-free/Ca2+ reintroduction method. As shown in Fig. 5A, in a Ca2+-free KHB buffer containing 0.5 mM EGTA, thapsigargin (2 µM) produced a transient increase in [Ca2+]i, from 60 to 120 nM, which slowly declined to resting levels after 3 min. Reintroduction of 1.28 mM Ca2+ caused a dramatic increase in [Ca2+]i that was blocked by La3+ (50 µM). In parallel cAMP time course experiments, the simultaneous addition of isoproterenol plus thapsigargin (2 µM) in a Ca2+-free KHB buffer containing 0.5 mM EGTA did not increase cAMP at any of the time periods examined (Fig. 5B); cAMP levels were similar to those observed with isoproterenol alone. On the introduction of 1.28 mM Ca2+ to the incubation buffer at 1 min, however, there was a significant sustained increase in cAMP accumulation that was blocked by La3+ (50 µM).


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  		Fig. 5.   Effects of thapsigargin on [Ca2+]i and stimulated cAMP accumulation in mouse parotid acini in a Ca2+-free KHB buffer. A: thapsigargin (2 µM) was added at 60 s and 1.28 mM Ca2+ was added at 480 s (trace a). In trace b, La3+ (50 µM) was added 2 min before addition of 1.28 mM Ca2+. B: isoproterenol (0.1 µM) + thapsigargin (2 µM) were added for 1 min in absence of Ca2+, followed by reintroduction of 1.28 mM Ca2+ (trace a), or no addition of Ca2+ (trace b), or reintroduction of 1.28 mM Ca2+ in presence of La3+ (50 µM; trace c). Results are representative of 3 experiments.

Relationship between ionomycin augmentation of isoproterenol-stimulated cAMP accumulation and [Ca2+]i. The Ca2+ ionophore ionomycin was also used to manipulate [Ca2+]i and, like thapsigargin, acts independently of receptor activation and inositol trisphosphate formation. Ionomycin was used at a concentration that is somewhat selective for intracellular membranes (18). Data presented in Fig. 6 show that, in a KHB buffer containing 1.28 mM Ca2+, ionomycin (0.5 µM) mimicked the effects of carbachol on cAMP accumulation. The ionomycin response was similar in onset to that produced by carbachol.


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  		Fig. 6.   Time course of cAMP accumulation in mouse parotid acini stimulated by isoproterenol (0.1 µM) and by isoproterenol + ionomycin (Iono, 0.5 µM) in a Ca2+ (1.28 mM)-containing KHB buffer. Results are representative of 5 experiments.

The relationships between Ca2+ release, capacitative Ca2+ entry, and cAMP accumulation are shown in Fig. 7. In a Ca2+-free KHB buffer containing 0.5 mM EGTA, ionomcycin, like thapsigargin, produced a transient increase in [Ca2+]i, from 70 to 118 nM, which slowly declined to resting levels (Fig. 7A). Reintroduction of 1.28 mM Ca2+ at 8 min caused a dramatic increase in [Ca2+]i that was blocked by La3+ (50 µM). In parallel cAMP time course experiments, the addition of isoproterenol plus ionomycin in a Ca2+-free KHB buffer containing 0.5 mM EGTA did not increase cAMP at any of the time periods examined (Fig. 7B); cAMP levels were similar to those observed with isoproterenol alone. On reintroduction of 1.28 mM Ca2+ at 1 min, however, there was a significant sustained increase in cAMP accumulation that was blocked by La3+ (50 µM).


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  		Fig. 7.   Effects of ionomycin on [Ca2+]i and stimulated cAMP accumulation in mouse parotid acini in a Ca2+-free KHB buffer. A: ionomycin (0.5 µM) was added at 60 s and 1.28 mM Ca2+ was added at 480 s (trace a). In trace b, La3+ (50 µM) was added 2 min before addition of 1.28 mM Ca2+. B: isoproterenol (0.1 µM) + ionomycin (0.5 µM) were added for 1 min in absence of Ca2+, followed by reintroduction of 1.28 mM Ca2+ (trace a), no addition of Ca2+ (trace b), or reintroduction of 1.28 mM Ca2+ in presence of La3+ (50 µM; trace c). Results are representative of 3 experiments.

Involvement of enzymes that synthesize and degrade cAMP in muscarinic augmentation of stimulated cAMP levels. Because the above augmentation studies were conducted in the absence of a PDE inhibitor, the steady-state levels of cAMP measured represent the net effect of changes in the enzymes that both synthesize and degrade cAMP. Further experiments were designed to evaluate whether augmentation of stimulated cAMP accumulation results from inhibition of a PDE isoenzyme or activation of AC isoenzyme(s). For the studies presented, acini were treated with and without the nonspecific PDE inhibitor IBMX (100 µM) and milrinone (10 µM) for 10 min, in the absence and presence of 1.28 mM Ca2+, before the addition of isoproterenol plus carbachol, or isoproterenol plus thapsigargin. The rationale for using milrinone, a specific inhibitor of the guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited PDE (cGI) (20), was based on previous findings supporting a role for cGMP in muscarinic augmentation of forskolin-stimulated cAMP accumulation (30).

It was expected that, if augmentation was due to inhibition of a PDE isoenzyme, then carbachol would not augment isoproterenol-stimulated cAMP levels in the presence of IBMX. As shown in Fig. 8A, time course studies showed that, in the absence of IBMX and in a Ca2+-free buffer, carbachol plus isoproterenol-stimulated cAMP accumulation was less than in Ca2+-containing KHB buffer, due to activation of a PDE isoenzyme (see Ref. 28; also see Fig. 1); cAMP values were 112.0 and 220.6 pmol/mg protein, respectively, at 5 min. When IBMX (100 µM) was added to the Ca2+-free KHB buffer, cAMP levels increased dramatically to 1,089.6 pmol/mg protein at 5 min. Values obtained with isoproterenol alone in the absence and presence of IBMX were 141.0 ± 18 and 830.5 ± 33 pmol/mg protein, respectively (Table 1).


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  		Fig. 8.   Time course of effects of 3-isobutyl-1-methylxanthine (IBMX; 100 µM) on carbachol (A) and thapsigargin (B) augmentation of isoproterenol-stimulated cAMP accumulation. Acini were preincubated with the phosphodiesterase inhibitor for 10 min, in absence and presence of 1.28 mM Ca2+, before addition of isoproterenol (0.1 µM) + carbachol (10 µM) or isoproterenol + thapsigargin (2 µM). Results are representative of 5 experiments.

                              
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  		Table 1.   Effects of the phosphodiesterase inhibitors IBMX and milrinone on augmentation of stimulated cAMP accumulation in mouse parotid acini

In a Ca2+-containing buffer containing IBMX (100 µM), isoproterenol plus carbachol further increased cAMP levels to 1,726.4 pmol/mg protein. cAMP levels in the presence of isoproterenol alone were 130.2 ± 17 and 902.9 ± 56 pmol/mg protein, respectively (Table 1). The effects of IBMX on thapsigargin and ionomycin augmentation of stimulated cAMP levels in the absence and presence of Ca2+ were similar to those produced by carbachol (Fig. 8B and Table 1).

If cGMP were involved in muscarinic augmentation of stimulated cAMP levels by activating cGI, then it would be expected that milrinone would mimic the effects of carbachol in augmenting stimulated cAMP levels. Incubation of acini for 10 min with milrinone (10 µM) did not modify isoproterenol-stimulated cAMP accumulation. At 5 min, isoproterenol-stimulated values were 132.2 ± 18.4 and 129.0 ± 14.5 pmol/mg protein in the absence and presence of milrinone, respectively. Also, as shown in Table 1, preincubation of acini with milrinone had no effect on carbachol augmentation of isoproterenol-stimulated cAMP levels in a Ca2+-free or Ca2+-containing buffer (Table 1).

Of the three ACs stimulated by Ca2+ in vitro (2, 5, 25), ACI and ACVIII have been reported to be exclusively regulated by capacitative Ca2+ entry (9, 10). It was noted that in HEK-293 cells transfected with ACVIII, carbachol-induced Ca2+ release was also found to stimulate AC (10) in a manner and time frame similar to those observed in mouse parotid acini. Furthermore, the stimulatory effect on AC was dependent on emptying of Ca2+ stores, as thapsigargin eliminated the response. Based on these findings, experiments were conducted to determine whether ACVIII is expressed in mouse parotid glands. In Fig. 9, poly(A)+-enriched mouse parotid and neocortex RNA were analyzed by blot hybridization using a cDNA probe specific for ACVIII. Two distinct type VIII messages of 5.5 and 4.4 kilobases were observed for both tissues.


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  		Fig. 9.   Northern blot analysis of type VIII adenylyl cyclase mRNA in mouse parotid gland (P) and neocortex (N). RNA (2 µg) was loaded in each lane and Northern blot analysis was carried out as described in MATERIALS AND METHODS. Kb, kilobase.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, the time course of stimulated cAMP accumulation was examined to provide a useful approach for determining the importance of capacitative Ca2+ entry and Ca2+ release from intracellular stores in agonist augmentation of cAMP accumulation in mouse parotid acini. The important findings of this study were that 1) capacitative Ca2+ entry is sufficient to produce the sustained effects of carbachol, thapsigargin, and ionomycin on stimulated cAMP accumulation; 2) Ca2+ release from intracellular stores, although insufficient to affect cAMP levels by thapsigargin and ionomycin, appears to be required for the early rapid phase of muscarinic augmentation of stimulated cAMP accumulation; and 3) Ca2+ appears to play a role in promoting AC synthesis.

An involvement of capacitative Ca2+ entry in cAMP metabolism has been reported for C6-2B glioma cells (3), SH-Sy5y human neuroblastoma cells (14), and HEK-293 cells transfected with ACI and ACVIII (9, 10, 31). In C6-2B glioma cells, thapsigargin, ionomycin, and UTP, agents that affect intracellular Ca2+ release by different mechanisms, increased Ca2+ entry, which was associated with inhibition of stimulated cAMP synthesis (3), whereas, in HEK-293 cells transfected with ACI and ACVIII, capacitative Ca2+ entry was associated with augmentation of stimulated cAMP synthesis (9, 10). Capacitative Ca2+ entry also appears to be associated with augmentation of isoproterenol-stimulated cAMP synthesis in mouse parotid acini, as augmentation was observed in the presence of IBMX at concentrations sufficient to inhibit soluble PDE activity.

Of interest was the finding that, although thapsigargin and/or ionomycin did not affect stimulated cAMP accumulation in mouse parotid acini, or cAMP levels in HEK-293 cells transfected with ACI and ACVIII in a Ca2+-free buffer (10, 31), carbachol was found to augment stimulated cAMP accumulation at early time points in mouse parotid acini and in HEK cells expressing ACVIII (10). In addition, carbachol alone also increased cAMP levels at early time periods in SH-Sy5y cells under conditions where Ca2+ influx was eliminated (14). Time course studies of cAMP accumulation also revealed that the presence of a PDE inhibitor was required for the detection of carbachol-stimulated cAMP levels at early time periods in HEK-293 cells expressing ACVIII and in SH-Sy5y and mouse parotid acini. Carbachol (1 mM), in the presence of IBMX (1 mM), increased cAMP levels by ~40% at 0.5 min in SH-Sy5y cells (14) and by 70% at 1 min in HEK-293 cells incubated with 0.5 mM IBMX. In mouse parotid acini, in the presence of IBMX (100 µM), carbachol (10 µM) increased cAMP levels twofold (unpublished observations).

To date, nine different isoforms of adenylate cyclase have been cloned; their existence suggests that they may be differentially regulated. The enzymes exhibit type-specific stimulatory and inhibitory regulation by G protein alpha - and beta gamma -subunits, Ca2+, calmodulin, forskolin, P-site inhibitors, and PKC (15, 22, 37). A number of the members of the family of ACs can be regulated by alterations in intracellular Ca2+. Of these, ACI, ACIII, and ACVIII are stimulated by Ca2+ in vitro (2, 6, 25). In vivo, ACI and ACVIII are stimulated and ACIII is inhibited by Ca2+ (2, 5, 33, 35). Capacitative Ca2+ entry has been shown to be sufficient for stimulation of ACI and ACVIII (9, 10). Our present finding of ACVIII expression in mouse parotid acini, combined with previous findings that Ca2+ stimulates AC and augments the effects of forskolin on cyclase activity in membrane fractions and intact cells (21, 30), is consistent with results obtained in HEK-293 cells expressing ACVIII (2, 10). Furthermore, Ca2+ in combination with agonists acting via the G protein subunit alpha s produced a synergistic stimulation of cAMP accumulation in parotid cells and in HEK cells expressing ACVIII. Although ACVIII has only been reported to be present in brain (2), this represents only one study in which a limited number of tissues, excluding parotid, were examined. Furthermore, recent studies using multiple primers and both polymerase chain reaction and Northern blot analysis show that ACVIII is present in non-brain tissues, including heart and kidney (L. Muglia, personal communication). Although both ACVIII and ACI can be stimulated by capacitative Ca2+ entry (9, 10, 31) and ACI can be activated in a similar fashion to ACVIII (5, 33), it is unlikely that ACI is also expressed in mouse parotid acini and that it plays a role in cAMP metabolism, since this AC isoform appears to be specifically expressed in neural tissues (36).

The finding that isoproterenol stimulates cAMP in mouse parotid acini but has little or no effect on cAMP levels in HEK-293 cells transfected with either ACI or ACVIII (2, 33) is paradoxical, as Gsalpha clearly stimulates AC and produces a synergistic effect with Ca2+/calmodulin in isolated membrane fractions (2). The fact that isoproterenol alone stimulates AC activity in mouse parotid acini suggests that another AC isoenzyme is involved in regulated cAMP synthesis. A likely candidate is ACIII, as this isoenzyme has been shown to be activated by isoproterenol without increasing intracellular Ca2+ in HEK-293 cells expressing ACIII (10, 34).

The ability of carbachol, but not thapsigargin and ionomycin, to augment stimulated cAMP accumulation at early time periods in mouse parotid acini, SH-Sy5y (14), and HEK-293 cells expressing ACVIII (10) suggests that the release of Ca2+ per se from intracellular stores is insufficient to affect cAMP levels but appears to be required for this response. This is supported by the present data as well as data from HEK-293 cells expressing ACVIII (10), showing that pretreatment of cells with thapsigargin eliminated this effect. The fact that beta gamma -subunits of G proteins as well as PKC, generated as a consequence of receptor interaction, have specific effects on AC isoenzymes (15, 16, 31) suggests that AC isoenzymes other than ACVIII may contribute to the augmentation effects of carbachol on stimulated cAMP accumulation at early time periods in a Ca2+-free buffer. To date, there is no evidence of beta gamma -subunit activation of AC isoenymes that are known to be stimulated by Ca2+.

On the other hand, there is support for an involvement of PKC in enzyme activation. Watson et al. (32) showed that treatment of HEK-293 cells, overexpressing ACVII, with phorbol ester resulted in a synergistic increase in beta -adrenergic-stimulated cAMP accumulation. This increase was noted at early time periods and peaked by 4 min. Phorbol esters have also been found to enhance forskolin responsiveness of ACI in HEK-293 cells (4). Because we reported previously that PKC can stimulate AC and augment the effects of isoproterenol in isolated membrane fractions (29), it is suggested that ACVII or ACI may be involved in the early rapid phase of stimulated cAMP accumulation. The observation that increases in intracellular Ca2+ failed to result in increases in cAMP levels in ACVII-expressing cells (31) is consistent with data showing that intracellular Ca2+ release alone is insufficient to activate cAMP in mouse parotid acini. Activation of the type II isoform by PKC, reported to occur more gradually in HEK-293 cells with peak activity at 20 min, however, would not be consistent with the time frame of cAMP accumulation noted herein for mouse parotid acini. Further studies are required to determine whether these AC isoenzymes are present in mouse parotid cells.

In summary, by using identical protocols to examine the relationship between agonist-induced capacitative Ca2+ entry and augmentation of stimulated cAMP accumulation, time course studies revealed that the two events are coincident. Data also suggest that augmentation is due to increased cAMP synthesis rather than to decreased PDE activity and may involve multiple AC isoenzymes. The lack of effect of thapsigargin and ionomycin on cAMP levels in a Ca2+-free media, despite a substantial release of intracellular Ca2+, is consistent with the hypothesis that Ca2+-stimulated ACs are compartmentalized in the same domain as are capacitative Ca2+ entry channels and thus are functionally colocalized for cAMP synthesis to be regulated by Ca2+ (3, 10).

    ACKNOWLEDGEMENTS

We thank Scott Young for helpful comments.

    FOOTNOTES

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

Address for reprint requests: E. L. Watson, Dept. of Oral Biology. Box 357132, Univ. of Washington, Seattle, WA 98195-7132.

Received 17 December 1996; accepted in final form 15 December 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(3):C557-C565
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society


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