Stimulation of Cyclic ADP-ribose Synthesis by Acetylcholine and Its Role in Catecholamine Release in Bovine Adrenal Chromaffin Cells*

(Received for publication, October 11, 1996, and in revised form, May 15, 1997)

Katsuya Morita , Shigeo Kitayama and Toshihiro Dohi Dagger

From the Department of Pharmacology, Hiroshima University School of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Cyclic ADP-ribose (cADPR) is suggested to be a novel messenger of ryanodine receptors in various cellular systems. However, the regulation of its synthesis in response to cell stimulation and its functional roles are still unclear. We examined the physiological relevance of cADPR to the messenger role in stimulation-secretion coupling in cultured bovine adrenal chromaffin cells. Sensitization of Ca2+-induced Ca2+ release (CICR) and stimulation of catecholamine release by cADPR in permeabilized cells were demonstrated along with the contribution of CICR to intracellular Ca2+ dynamics and secretory response during stimulation of intact chromaffin cells. ADP-ribosyl cyclase was activated in the membrane preparation from chromaffin cells stimulated with acetylcholine (ACh), excess KCl depolarization, and 8-bromo-cyclic-AMP. ACh-induced activation of ADP-ribosyl cyclase was dependent on the influx of Ca2+ into cells and on the activation of cyclic AMP-dependent protein kinase. These and previous findings that ACh activates adenylate cyclase by Ca2+ influx in chromaffin cells suggested that ACh induces activation of ADP-ribosyl cyclase through Ca2+ influx and cyclic AMP-mediated pathways. These results provide evidence that the synthesis of cADPR is regulated by cell stimulation, and the cADPR/CICR pathway forms a significant signal transduction for secretion.


INTRODUCTION

An elevation of cytosolic free Ca2+ concentrations ([Ca2+]i)1 is a prerequisite for cellular functions of numerous types of cells, including neurons. Influx of Ca2+ through plasma membrane pathways and mobilization of Ca2+ from intracellular stores by inositol 1,4,5-trisphosphate (IP3), a second messenger derived from membrane phosphoinositide, are well documented mechanisms for increase in [Ca2+]i.

Recent attention has been focused on another mechanism for mobilizing intracellular Ca2+: Ca2+-induced Ca2+ release (CICR), mediated by the ryanodine receptors. CICR mediates the amplification and propagation of initial Ca2+ signals, the generation of Ca2+ oscillations, and the propagation of Ca2+ waves in certain types of cells. Depolarizing stimuli can activate release of Ca2+ from ryanodine-sensitive intracellular stores in a number of neuronal cells (1). In cerebellar granule cells, a major component of both K+ depolarization- and N-methyl-D-aspartate-induced elevation of [Ca2+]i appears to be due to the release of Ca2+ from intracellular stores, evidence that suggests the importance of intracellular Ca2+ release via CICR (2).

Galione et al. (3) recently identified a novel Ca2+ mobilizing agent that is a cyclic metabolite of nicotinamide adenine dinucleotide (NAD+), cyclic ADP-ribose (cADPR). This agent is as potent as IP3 in mobilizing Ca2+ in sea urchin eggs and mediates the fertilization-induced Ca2+ wave in these eggs (3, 4). The presence of cADPR and the enzyme-catalyzing conversion of NAD+ into cADPR and the ability of cADPR to release Ca2+ through an IP3-insensitive mechanism have been demonstrated in many tissues (5-18). cADPR has been shown to activate the cardiac but not the skeletal isoforms of the ryanodine receptor Ca2+ channel and could be a candidate for a nonskeletal type ryanodine receptor endogenous messenger (8). If cADPR plays a role as a second messenger of CICR, it is necessary to demonstrate that its intracellular levels are under the control of extracellular stimuli. Recently, Takasawa et al. (7) suggested that cADPR is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization and insulin release. However, little evidence is available in this regard for various cell types. In addition, recent studies using type 2 cardiac ryanodine receptors incorporated into planar lipid bilayers have shown the inability of cADPR to cause Ca2+ release in the presence of physiological concentrations of ATP, indicating that cADPR is unlikely to be a second messenger for CICR in vivo (19, 20). In addition, Higashida et al. (21) suggest another role of cADPR, that it may mediate the muscarinic acetylcholine (ACh) receptor-induced inhibition of M-type K+ currents in NG108-15. Therefore, the physiological relevance of cADPR remains unclear.

Adrenal medullary chromaffin cells are widely used as a model for the analysis of endocrine and neuronal cell functions. Caffeine is well known for inducing a large increase in [Ca2+]i levels through the mobilization of Ca2+ from intracellular Ca2+ stores by stimulating CICR in adrenal chromaffin cells. Spontaneous [Ca2+]i fluctuations in rat chromaffin cells are generated by caffeine (22). Thus the presence of a ryanodine-sensitive intracellular Ca2+ store is suggested. Here, we demonstrate the ability of cADPR to cause Ca2+ release and the activation of cADPR synthesis in response to stimuli in bovine adrenal chromaffin cells, the results suggesting that the Ca2+ mobilizing pathway mediated by cADPR may participate in physiological stimulation-induced secretory response of the cells.


EXPERIMENTAL PROCEDURES

Materials

Cyclic ADP-ribose was purchased from Amersham International Public Limited Company (Buckinghamshire, UK) and Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Inositol 1,4,5-trisphosphate, fura-2, and fura-2/AM were obtained from Dojindo Laboratories (Kumamoto, Japan); collagenase-S1, from Nittagelatin, Inc. (Osaka, Japan); thapsigargin, from LC Services Corporation (Woburn, MA); ryanodine, from AgriSystems International (Wind Gap, PA); Calcium SpongeTM S (BAPTA conjugated with polystyrene), from Molecular Probes, Inc. (Eugene, OR); imperatoxin inhibitor, from Latoxan (Rosans, France); cyclic GDP-ribose, nicotinamide guanine dinucleotide (NGD+), and the catalytic subunit of cyclic AMP-dependent protein kinase and its inhibitor, from Sigma; cAMP-S, Rp-diastereomer (Rp-cAMP-S) sodium salt, from BioLog Life Science Institute (Bremen, Germany); H-89, from Seikagaku-Kogyo (Tokyo, Japan); [2,5',8-3H]cyclic ADP-ribose (1.29 TBq/mmol), from Amersham; 45CaCl2 (1.55-1.13 GBq/mg of Ca) and [adenylate-32P]NAD+ (29.6 TBq/mmol), from NEN Life Science Products. Other chemicals, including caffeine and digitonin, were obtained from Wako and Sigma.

Cell Preparation and Culture

Chromaffin cells of bovine adrenal glands were isolated enzymatically according to the procedure described by Fenwick et al. (23), with some modifications (24, 25). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin G (100 units/ml), streptomycin (100 µg/ml), ascorbate (0.1 mM), and HEPES (5 mM) for 24-72 h at 37 °C under 5% CO2, 95% air as suspension culture for measurements of Ca2+ release and [Ca2+]i or as monolayer culture on 35-100-mm tissue culture dish (5 × 105 cells/ml) for 2-5 days for catecholamine (CA) release assay. Cells were washed and suspended before use in a medium containing NaCl (150 mM), KCl (5 mM), MgSO4 (1 mM), CaCl2 (1.3 mM), glucose (5 mM), HEPES-Tris buffer (10 mM), and bovine serum albumin 0.5%, pH 7.4.

Measurements of Ca2+ Release and [Ca2+]i

For measurement of Ca2+ release from digitonin-permeabilized chromaffin cells, cells were washed and suspended in potassium glutamate buffer (145 mM potassium glutamate, 20 mM PIPES, 1 mM EGTA, pH 6.6) containing an ATP generating system (2 mM Mg2+-ATP, 5 mM creatine phosphate, 40 units/ml creatine phosphokinase) and protease inhibitors (2.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 50 µg/ml trypsin inhibitor) and then given permeabilization by incubating cells with digitonin (20 µM) for 5 min at 25 °C. The cells were washed and resuspended (107 cells/ml) in an intracellular medium (KH medium: 140 mM KCl, 10 mM NaCl, 30 mM HEPES, pH 7.0) containing an ATP generating system, protease inhibitors, mitochondrial inhibitors (10 µg/ml antimycin A, 10 µg/ml oligomycin, and 10 mMNaN3), and 0.025% bovine serum albumin. The permeabilization was checked by measuring the leakage of lactate dehydrogenase. One milliliter of cell suspension was transferred to a fluorescence cuvette and supplemented with fura-2 (1 µM). Fluorescence was continuously monitored using a fluorometer at an excitation of 340/380 nm and an emission of 510 nm. Increase in Ca2+ concentration in the medium was calibrated by the addition of known amounts of Ca2+ and expressed as Ca2+ release in the text.

For the measurement of [Ca2+]i, cells were incubated at 32 °C with 1 µM fura-2 AM for 30 min in order to load the dye. Cells were then centrifuged at 15 × g for 10 min and resuspended to yield 3 × 106 cells/ml. Fluorescence was measured using a dual-wavelength, fluorescence spectrophotometric mode with an excitation of 340 and 380 nm and an emission of 510 nm. [Ca2+]i was calculated from the fluorescent ratio at 340 and 380 nm using the equation of Grynkiewicz et al. (26) and a value of 224 nM for the Kd of fura-2.

For assaying 45Ca2+ release, a monolayer culture of chromaffin cells was preincubated in the full culture medium containing 45CaCl2 (185 kBq/ml) for 24-48 h in the CO2 incubator. Then cells were washed and permeabilized as described above. After a 1-min preincubation in a KH medium containing an ATP generating system, protease inhibitors, mitochondrial inhibitors, and 1 mM EGTA at 32 °C, cells were incubated in various conditions. The medium was then immediately separated from the cells, and the 45Ca2+ released into the medium was quantified by liquid scintillation counting.

Measurement of CA Release

For measurement of CA release from permeabilized chromaffin cells, a monolayer culture of chromaffin cells was permeabilized as described above, preincubated for 1 min in a KH medium containing an ATP generating system, protease inhibitors, and mitochondrial inhibitors with or without 1 mM EGTA, and then incubated for 20 min with cADPR or IP3. After the period of incubation, the medium was separated from the cells and used for the CA assay. For this experiment, the KH medium was treated with Calcium SpongeTM S before use to reduce the background of CA released due to contaminated Ca2+ in the medium. Various concentrations of Ca2+ in the medium were made by a Ca2+-EGTA buffer, and the free Ca2+ concentrations were measured using a selective Ca2+ minielectrode or specific dye indicators, fura-2 and fluo 3. Perchloric acid (5% of final concentration) was added to the incubation medium, which was then centrifuged at 4,500 × g for 15 min. 0.2 ml of the clear supernatant was diluted 20-fold with 3 M acetate buffer to create an appropriate concentration of CA and to adjust the pH to 6.2. Total CA in the medium was determined fluorometrically by the trihydroxyindole method (27), with adrenaline serving as a standard. CA release from intact chromaffin cells was performed as described previously (24, 25).

Assay of ADP-ribosyl Cyclase

Cultured chromaffin cells were incubated in various conditions and then washed rapidly twice with 10 ml of chilled buffer containing 0.34 M glucose, 1 mM MgCl2, 10 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml soybean trypsin inhibitor, 20 mM HEPES, pH 7.2, and immersed in liquid N2. Cells were homogenized in the buffer using Polytron for 20 s followed by six strokes with a Thomas homogenizer equipped with Teflon pestle. The homogenate was centrifuged at 100,000 × g for 30 min at 0 °C. The resulting pellet was suspended in the same buffer as a membrane fraction. The homogenate and membrane fraction were then used for assays of enzyme activity and protein concentration.

ADP-ribosyl cyclase activity was determined by measuring the production of cADPR from [32P]NAD+ as substrate. An assay mixture containing 10 µl of enzyme preparation, 40 µl of 250 µM [32P]NAD+ (37 kBq), 20 mM HEPES, pH 7.2, and 0.1% Triton X-100 was incubated at 37 °C for 10 min. The resulting [32P]cADPR was separated from substrate and metabolites by selective hydrolysis with snake venom phosphodiesterase 1 followed by purification with dihydroxyboronyl Bio-Rex 70 (DHB Bio-Rex 70) column chromatography according to the reported procedure (28). In brief, the reaction mixture was treated with snake venom phosphodiesterase 1 (0.3 unit), diluted with ammonium formate buffer, pH 9.0, and applied to a DHB Bio-Rex 70 column. After washing the column with the above buffer, [32P]cADPR was eluted with 5 ml of deionized water and quantified by liquid scintillation counting to estimate cyclase activity. Eighty ml of eluant from the DHB Bio-Rex 70 column was neutralized, freeze-dried, and then dissolved with 200 µl of deionized water. Further analysis of the sample by HPLC was performed using a TSKgel QAE-2SW column (0.46 × 25 cm) and TSKgel ODS-80Ts column (0.46 × 15 cm) connected in tandem. Elution was performed with 0.25 M ammonium formate buffer, pH 4.0, at a flow rate of 0.5 ml/min and monitored by the absorbance at 260 nm. One-ml fractions were assayed for determination of Ca2+ release activity as described above and for radioactivity.

The activity of ADP-ribosyl cyclase was also determined by measuring cyclic GDP-ribose (cGDPR) fluorometrically using NGD+ as a substrate (29). cGDPR is resistant to hydrolysis so this procedure was demonstrated to be highly sensitive and convenient for ADP-ribosyl cyclase-like enzymes such as CD38 (29). Reaction mixtures containing 60 µM NGD+, 20 mM Tris, pH 7.4, and homogenate and membranes of chromaffin cells (0.8 mg of protein/ml) were maintained at 37 °C for 1 h to continuously monitor the fluorescence of cGDPR on a Hitachi 850 spectrophotometer at excitation/emission wavelengths of 300/410 nm. The fluorescence intensity was calibrated and converted to molar concentration by use of authentic cGDPR. NGD+ was HPLC-purified according to the procedure of Graeff et al. (29).

Assay of cADPR Hydrolase Activity

The hydrolase activity was determined using 200 µM [3H]cADPR (7.4 kBq) as a substrate as reported (28). ADPR, the product of cADPR was first converted to AMP by treatment with phosphodiesterase, and the AMP was separated on the DHB Bio-Rex 70 column. The flow-through and wash fractions (4 ml) were combined, and [3H]AMP was quantitated by liquid scintillation counting. Statistical analyses were performed with the Student's t test.


RESULTS

Characterization of cADPR-induced Ca2+ Release from Digitonin-permeabilized Chromaffin Cells

cADPR stimulated Ca2+ release in a concentration-dependent manner from permeabilized chromaffin cells with larger maximal response and lower affinity than that induced by IP3 (Fig. 1A). In the presence of 1 µM IP3, in which IP3 caused the maximal release, additional 1 µM IP3 caused no further increase in Ca2+ release, whereas cADPR brought about further increase in the release. As cADPR may induce Ca2+ release by different mechanisms than that induced by IP3, we first examined the pharmacological characteristics of cADPR-induced Ca2+ release in chromaffin cells.


Fig. 1. cADPR- and IP3-induced Ca2+ release from digitonin-permeabilized bovine adrenal chromaffin cells. A, concentration-response curves of cADPR- and IP3-induced Ca2+ release. The solid triangle represents the addition of 0.5 µM cADPR, and the open triangle represents the addition of 1 µM IP3 in the presence of 1 µM IP3. The solid lines represent the best fit to the data. Values are the mean ± S.E. of the peak rise of Ca2+ release (n = 5-18). B, typical patterns of Ca2+ release induced by cADPR (a) and IP3 (d) and the effects of 8-Br-cAMP (b and f) and calmodulin (CaM, c and e) on the Ca2+ release. Each trace of Ca2+ release was determined with samples of the same cell preparation. 8-Br-cAMP (0.3 mM) and calmodulin (1 µM) were added 3 min before the addition of cADPR (0.3 µM) or IP3 (0.3 µM). These and the following experiments are representative examples of experiments performed with very similar results in at least three different batches of cells.
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Phosphorylation of the ryanodine receptor by calmodulin-dependent protein kinase has been reported for sarcoplasmic reticulum from cardiac muscle (30, 31), where calmodulin inhibits the channel activity (31). Phosphorylation of ryanodine receptor and IP3 receptor by cAMP-dependent protein kinase (A-PK) also has been reported, although the functional role of the phosphorylation in the regulation of Ca2+ release is not clear (30-35). In the present study, 8-bromo-cyclic-AMP (8-Br-cAMP) potentiated both cADPR- and IP3-induced Ca2+ release; calmodulin potentiated cADPR-induced release but did not affect IP3-induced release (Fig. 1B). The catalytic subunit of A-PK (A-PK C.S.) potentiated cADPR- and IP3-induced Ca2+ release, and the potentiating effects of an A-PK C.S. were blocked by the A-PK inhibitor (Table I). 8-Bromo-cyclic-GMP (8-Br-cGMP) was without effect (data not shown). A calmodulin antagonist, W-7, reduced cADPR-induced release and blocked the potentiations by calmodulin of cADPR- and caffeine-induced Ca2+ release. Neither calmodulin nor W-7 affected IP3-induced Ca2+ release.

Table I. Effects of various treatments on Ca2+ release induced by cADPR, caffeine, ryanodine, and IP3

IpTxi was treated 30 min before the addition of Ca2+ releasers. Other drugs were treated 1-3 min before the addition of Ca2+ releasers. Values are the mean ± S.E. of the peak rise of Ca2+ release (n = 4-8).

Addition Increase in Ca2+ concentration
cADPR (0.3 µM) Caffeine (0.5 mM) Ryanodine (1 µM) IP3 (0.3 µM)

µM
Nonea 0.12  ± 0.01 0.42  ± 0.02
A-PK C.S. (200 units) 0.26  ± 0.03b 0.67  ± 0.06b
A-PK inhibitor (1200 units) 0.09  ± 0.01 0.43  ± 0.03
A-PK inhibitor + A-PK C.S. 0.10  ± 0.02 0.42  ± 0.03
None 0.17  ± 0.03 0.15  ± 0.02 0.82  ± 0.06
Calmodulin (1 µM) 0.31  ± 0.07b 0.26  ± 0.05b 0.79  ± 0.09
W-7 (100 µM) 0.04  ± 0.01c 0.11  ± 0.02b 0.72  ± 0.06
W-7 + calmodulin 0.02  ± 0.00c 0.14  ± 0.03 0.70  ± 0.03
None 0.32  ± 0.03 0.51  ± 0.04
Heparin (30 µg/ml) 0.29  ± 0.03 0.02  ± 0.01c
CsCl (10 mM) 0.33  ± 0.03 0.05  ± 0.02c
Tetraethylammonium (30 mM) 0.29  ± 0.04 0.04  ± 0.02c
Benzocaine (0.3 mM) 0.08  ± 0.01c 0.48  ± 0.05
None 0.28  ± 0.04 0.19  ± 0.02 0.14  ± 0.03 1.17  ± 0.14
IpTxi (100 nM) 0.06  ± 0.03b 0.01  ± 0.01c 0.03  ± 0.01b 1.13  ± 0.16
None 0.05  ± 0.02 0.07  ± 0.01 0.50  ± 0.03
Caffeine (0.5 mM) 0.24  ± 0.04c 0.46  ± 0.02
cADPR (25 nM) 0.29  ± 0.04c 0.44  ± 0.01

a A-PK C.S. (46 units/µg of protein) was activated before use with 0.5 M DTT for 20 min at 32 °C and dialyzed for 3 h at 4 °C against the KH solution with 1 mM DTT. A-PK inhibitor was added directly to the KH solution containing 1 mM DTT. The control contained DTT at appropriate concentrations.
b p < 0.05, significantly different from control.
c p < 0.01, significantly different from control.

Heparin, CsCl, and tetraethylammonium, known inhibitors of IP3-induced Ca2+ release, all inhibited IP3-induced Ca2+ release in this experiment also but had no effect on cADPR-induced Ca2+ release. A classical inhibitor of ryanodine receptors, benzocaine, and a novel inhibitor, imperatoxin inhibitor (IpTxi), which has been purified from scorpion venom and shown to specifically block ryanodine receptors of skeletal and cardiac muscle (36), both, inhibited cADPR- but not IP3-induced release. Caffeine potentiated the effect of cADPR, and cADPR greatly potentiated caffeine-induced release. Such cross-potentiation was not observed between IP3 and cADPR or caffeine.

Thapsigargin, shown to specifically block the endoplasmic reticulum but not muscle sarcoplasmic reticulum Ca2+-ATPase, inhibits Ca2+ uptake into endoplasmic reticulum and then empties Ca2+ in the stores (37). In cells pretreated with 20 nM thapsigargin, IP3 failed to increase Ca2+ release. cADPR- and caffeine-induced Ca2+ release were only blocked by higher concentrations of thapsigargin (Fig. 2). The concentration-response relationship of thapsigargin-induced inhibition shows that Ca2+ release in response to IP3 is more sensitive to the inhibition than to cADPR. Taken together, these results may suggest that cADPR and caffeine act on the same Ca2+ release mechanism from the same stores, which are different from IP3-sensitive stores, and support the hypothesis that cADPR may work through a Ca2+ release system similar to the CICR system in the sarcoplasmic reticulum.


Fig. 2. Effects of thapsigargin (TG) on Ca2+ release induced by cADPR, IP3, and caffeine. Digitonin-permeabilized cells were incubated for 20 min with various concentrations of thapsigargin. The cells were then washed with thapsigargin-containing fresh medium by rapid centrifugation and resuspended immediately before the addition of Ca2+ releasers. The inset shows the concentration-response curves of thapsigargin-induced inhibition of cADPR (0.3 µM)- and IP3 (0.2 µM)-induced Ca2+ release. Values are the mean ± S.E. of the peak rise of Ca2+ release (n = 5-7).
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A number of studies on cADPR-induced Ca2+ release from sea urchin eggs have shown that the actions of ryanodine and caffeine are similar to those of cADPR (4, 38). Therefore, the ability of cADPR to sensitize CICR was examined by measuring Ca2+-induced 45Ca2+ release. Fig. 3 shows the effects of cADPR and caffeine on 45Ca2+ release from digitonin-permeabilized cells that had been prelabeled with 45CaCl2. cADPR stimulated 45Ca2+ release, with the peak effect occurring 10 s after the addition of cADPR. The effect of cADPR was transient in comparison with the long lasting release of 45Ca2+ induced by caffeine. cADPR and caffeine markedly enhanced 45Ca2+ release induced by low concentrations of Ca2+ but did not modify the maximal Ca2+-induced 45Ca2+ release.


Fig. 3. cADPR- and caffeine-induced stimulation of 45Ca2+ release. A, time course of 45Ca2+ release induced by cADPR and caffeine. Cells were prelabeled with 45Ca2+, permeabilized by digitonin, and then stimulated in the fresh medium containing 1 mM EGTA to adjust the pCa to 7.0. B, sensitization by cADPR and caffeine of Ca2+-induced 45Ca2+ release. The 45Ca2+ release was determined 5 s after the incubations in the medium with indicated stimulants, and pCa was obtained in Ca2+-EGTA buffer. 100 µM ryanodine + 10 mM caffeine (triangles) were also present during 45Ca2+ loading. The values represent the mean ± S.E. of five experiments assayed in quadruplicates.
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cADPR-induced CA Release

Increases in the concentration of Ca2+ in the medium from 0.02 µM to a peak effect of 3 µM stimulated release of CA in digitonin-permeabilized chromaffin cells (Fig. 4A). The Ca2+-induced CA release was not inhibited by IpTxi. In this cell preparation, cADPR (1 µM) caused an increase in CA release, and the effect of cADPR was blocked by EGTA and IpTxi (Fig. 4B). These results show the ability of cADPR to induce CA release via Ca2+ release through ryanodine receptor Ca2+ channel.


Fig. 4. Ca2+-induced CA release in digitonin-permeabilized chromaffin cells. A, relationship between free Ca2+ concentration in the medium and CA release and the effect of IpTxi. Following a 30-min pretreatment with (bullet ) or without (open circle ) 100 nM IpTxi, cells were permeabilized with digitonin. The medium was replaced with fresh medium containing the indicated concentrations of free Ca2+, and then the preparations were incubated for 20 min at 37 °C. After the incubation, the medium was separated from cells and saved for CA assay. B, cADPR-induced CA release and the inhibition by IpTxi and EGTA. Experimental designs are similar to those described in Fig. 4A, except that permeablized cells were washed with fresh medium before incubation. Values are the mean ± S.E. of five experiments assayed in triplicate; *, significantly different from the corresponding control at p < 0.01.
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Activation of ADP-ribosyl Cyclase and cADPR Hydrolase

To ascertain whether cADPR was a physiological messenger of CICR, the presence of synthesis and degradation systems for cADPR and their regulation by physiological cell stimulation was examined. Fig. 5 shows the presence of ADP-ribosyl cyclase activity in adrenal chromaffin cells. The product formed by the incubation of [32P]NAD+ with a homogenate of adrenal medulla was separated from the substrate and other metabolites by a selective hydrolysis with snake venom phosphodiesterase and then purified with cation-exchange chromatography, as reported previously (28). The incubation product was used for an assay of Ca2+ release from digitonin-permeabilized chromaffin cells. The product released Ca2+ in the IpTxi-sensitive manner (Fig. 5A). Identification of cADPR related to Ca2+-releasing activity in the product was further carried out using HPLC (Fig. 5, B, a). Elution profiles revealed a single peak at the same retention time as authentic cADPR with regard to both radioactivity and Ca2+-releasing activity. This procedure made it possible to assay ADP-ribosyl cyclase easily and accurately. Most cyclase activity was found in the centrifuged particulate fractions but was not detectable in the 120,000 × g supernatant (data not shown). In the homogenate prepared from the cells treated with ACh and excess KCl, ADP-ribosyl cyclase activity increased shortly after the additions (Fig. 5C). ACh-induced activation of the cyclase was further confirmed by HPLC. Both radioactivity (Fig. 5, B, b) and Ca2+-releasing activity (Fig. 5, B, c) were facilitated at the same retention time as authentic cADPR upon ACh-treated cells.


Fig. 5. Activation of ADP-ribosyl cyclase by the stimulation of chromaffin cells. A, release of Ca2+ by eluate from the DHB Bio-Rex 70 column of the reaction mixture of NAD+ and enzyme solutions with blocking by IpTxi. The eluate was neutralized, freeze-dried, and then dissolved in deionized water. The Ca2+ release activity of aliquots (a, b, and d) of eluate were examined in digitonin-permeabilized chromaffin cells. Treatment with IpTxi (100 nM) was carried out 30 min before permeabilization of the cells. B, aliquot of the eluant from the cells that were treated with or without ACh (30 µM) or from the sample without enzyme preparation was further analyzed by HPLC using a TSKgel QAE-2SW column and TSKgel ODS-80Ts column connected in tandem. Elution was performed with 0.25 M ammonium formate buffer, pH 4.0, and monitored by the absorbance at 260 nm as indicated by line with the positions of authentic compounds marked at the top (a). The elution from the HPLC column was collected in 1-ml fractions for quantitating the radioactivity (b) and for confirming the Ca2+-release activity (c). C, time courses of the activation of ADP-ribosyl cyclase in the cells treated with ACh (30 µM) and excess KCl (30 mM). After incubation with or without the indicated stimulants, cells were washed and homogenized with glucose-HEPES buffer containing protease inhibitors, then processed to the subsequent steps as described in (A) and quantified by liquid scintillation counting. D, cADPR hydrolase activity was determined as described under "Experimental Procedures." Values are the mean ± S.E. of three or four experiments assayed in duplicate.
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The presence of an enzyme that can degrade cADPR was noted during measurements of ADP-ribosyl cyclase activity in various tissue extracts. It has been reported that cADPR is degraded immediately by hydrolase and that the hydrolase activity, like ADP-ribosyl cyclase, is widely distributed (38), so we examined cADPR hydrolase activity in chromaffin cells. In the homogenate prepared from the cells treated with ACh (30 µM), cADPR hydrolase activity increased shortly after the addition of ACh (Fig. 5D). The extracts from both stimulated and nonstimulated cells were examined for their ability to promote Ca2+ release from digitonin-permeabilized cells. However no release of Ca2+ was detected in this assay system (data not shown). Thus the amount of cADPR might be less than that required for detectable release of Ca2+.

Detecting ADP-ribosyl cyclase activity was further performed by fluorometric assay of accumulated cGDPR from NGD+ as substrate instead of NAD+, which is resistant to hydrolysis (29). As shown in Fig. 6A, incubation of 60 µM NGD+ with homogenate prepared from chromaffin cells resulted in a progressive increase in the fluorescence, which can be calibrated using cGDPR as a standard. Identification of cGDPR as being responsible for the changes in fluorescence of the NGD+ products was further performed using HPLC. The fluorescent products had the same retention time as those obtained with authentic cGDPR (data not shown).


Fig. 6. Activation of ADP-ribosyl cyclase in chromaffin cells as measured by the conversion of NGD+ to cGDPR. A, homogenate prepared from adrenal chromaffin cells were incubated with 60 µM NGD+ in the presence of the ATP generating system, and the fluorescence of the resulting cGDPR was continuously monitored. 8-Br-cAMP (10 µM) and Rp-cAMP-S (0.5 mM) were added 1 and 10 min before the addition of NGD+, respectively. B, chromaffin cells were incubated for the indicated times with ACh (30 µM), forskolin (1 µM), and 8-Br-cAMP (0.1 mM), and then the membrane was prepared and measured as in A in the absence of an ATP generating system. Experiments were performed with very similar results on 3-8 different batches of cells, and typical results are shown. C, time courses of the activation of the enzyme in the cells treated with ACh (30 µM). Cells were treated with ACh for the various periods indicated and then fractionated to obtain membrane preparations. The enzyme activity was expressed as an amount of synthesized cGDPR at initial 2 min. Values are the means ± S.E. of eight experiments. D, effects of Ca2+ deprivation (Ca2+-defi.), diltiazem (10 µM), Rp-cAMP-S (0.5 mM), and H-89 (10 µM) on the activation of GDP-ribosyl cyclase by ACh (30 µM), forskolin (1 µM), and 8-Br-cAMP (0.1 mM). Cells were preincubated with diltiazem and the Ca2+-deficient medium in the presence of 0.1 mM EGTA for 5 min and Rp-cAMP-S and H-89 for 30 min before the incubation for 3 min with ACh, forskolin, or 8-Br-cAMP. Values are the mean ± S.E. of 3-8 experiments; *, significantly different from the corresponding control at p < 0.01.
[View Larger Version of this Image (38K GIF file)]

Mechanism of Receptor-mediated Activation of ADP-ribosyl Cyclase

The involvement of cAMP and calmodulin in the activation of ADP-ribosyl cyclase by ACh in intact cells was examined, since both agents enhanced cADPR-induced Ca2+ release in permeabilized cells (Fig. 1, Table I). In addition to the possibility of interaction of these agents with ryanodine receptor as suggested (32), another possible mechanism was an increase in cellular cADPR level by these agents. The cGDPR fluorescence actually increased linearly during the incubation of the homogenate preparation with 8-Br-cAMP in the presence of an ATP generating system. The rate of cGDPR synthesis increased from 77.0 ± 4.3 to 202.4 ± 11.1 pmol/mg of protein/min with the addition of 8-Br-cAMP, and the effect was blocked by a A-PK blocker, Rp-cAMP-S (Fig. 6A). Calmodulin was without effect (data not shown). In contrast to the observation shown in Fig. 6A, the membrane preparations from the ACh-treated cells revealed a rapid increase in fluorescence initially in the assay where ATP generating system was absent (Fig. 6B). Similar activation by forskolin and 8-Br-cAMP was observed (Fig. 6B). The activation of the enzyme by ACh was rapid, starting within 10 s and reaching a maximum at 1 min after the addition of ACh (Fig. 6C).

We have previously shown that ACh increased adenylate cyclase activity and accumulation of cAMP in chromaffin cells resulting from an influx of Ca2+ (24, 39). We therefore examined whether the activation of ADP-ribosyl cyclase by ACh is primarily mediated by Ca2+ influx. The action of ACh but not forskolin was abolished by the treatment of cells with Ca2+-free medium and an inhibitor of the voltage-operated Ca2+ channel (VOC), diltiazem (Fig. 6D). The treatments with A-PK blockers such as the structurally unrelated compounds H-89 and Rp-cAMP-S abolished the effect of both ACh and forskolin and also 8-Br-cAMP. These results strongly suggested that ADP-ribosyl cyclase was activated by ACh via Ca2+ influx and the resulting cAMP-mediated mechanism. It has been reported that cGMP can stimulate cADPR production in sea urchin eggs (40) and in PC12 cells (12). A similar mechanism responsible for cGMP does not seem to operate in chromaffin cells because sodium nitroprusside or 8-Br-cGMP did not affect the ADP-ribosyl cyclase activity in the same experimental protocol.2

Role of CICR in Secretory Response

Whether CICR participates in Ca2+ transience and CA release in chromaffin cells was examined using IpTxi. In digitonin-permeabilized chromaffin cells, IpTxi inhibited Ca2+ release in response to cADPR, caffeine, and ryanodine but not to IP3 (Table I). Pretreatment of intact chromaffin cells with IpTxi reduced ACh-induced [Ca2+]i increases (Fig. 7A).


Fig. 7. Effects of IpTxi and diltiazem on stimulation-induced [Ca2+]i increase in intact chromaffin cells. A, typical patterns of [Ca2+]i increase induced by ACh (5 µM) in the presence or absence of IpTxi (100 nM). t1/2 was then calculated from the time required for reduction of [Ca2+]i to half the peak high. B, relationship between the peak high of [Ca2+]i and t1/2 in various conditions. Ordinate values represent the increase in [Ca2+]i at peak highs induced by 5-15 µM ACh or 15-25 mM KCl in the presence or absence of IpTxi (100 nM) and diltiazem (1 µM). IpTxi and diltiazem were treated 30 and 5 min before the addition of ACh or excess KCl, respectively.
[View Larger Version of this Image (20K GIF file)]

As the increase in [Ca2+]i after stimulation of nicotinic ACh receptors in adrenal chromaffin cells is believed due to Ca2+ influx through its channel and VOC, the time required for the reduction of [Ca2+]i to half the peak high of [Ca2+]i (t1/2) was plotted as a function of [Ca2+]i obtained with various treatments. A linear correlation was found between t1/2 and [Ca2+]i in each condition (Fig. 7B). In the presence of diltiazem, the correlation between the peak high of the ACh-induced [Ca2+]i increase and t1/2 was very similar to that in the absence of diltiazem. However, t1/2 at the various concentrations of ACh in the presence of IpTxi was shortened, and the slope became steep. Similarly, the correlation between the peak high of excess KCl-induced [Ca2+]i increase and t1/2 was not modified by diltiazem but was shifted to the left by IpTxi. Thus, it is suggested that the attenuation of rise in [Ca2+]i is hastened by IpTxi through blockade of CICR. In other words, CICR contributes to a maintenance of the sustained [Ca2+]i rise during cell stimulation.

To further determine the involvement of CICR in CA release, we examined the effect of IpTxi on CA release that was induced by ACh in intact chromaffin cells. ACh at concentrations of 3-100 µM induced CA release with maximal effect at 30 µM. IpTxi reduced CA release induced by each concentration of ACh (Fig. 8A). Caffeine and ryanodine markedly potentiated ACh-induced CA release, and IpTxi blocked the potentiation by these compounds (Fig. 8B).


Fig. 8. Inhibition by IpTxi of ACh-induced CA release (A) and facilitation of ACh (5 µM)-induced CA release by ryanodine and caffeine (B). Cells were preincubated with medium containing IpTxi (100 nM) for 30 min before a 6-min stimulation with ACh. Ryanodine (1 µM) and caffeine (0.1 mM) were treated 3 min before the stimulation with ACh. CA release induced by ryanodine or caffeine alone was 0.02 ± 0.01 and 0.01 ± 0.01 µg/106 cells, respectively. Values are the mean ± S.E. of the net increase in CA release of 4-6 experiments assayed in triplicate; *, significantly different from the corresponding control at p < 0.01.
[View Larger Version of this Image (28K GIF file)]


DISCUSSION

A growing body of evidence supports the idea that cADPR might be an endogenous caffeine-like substance that regulates ryanodine receptor of type 2 in cardiac muscle and type 3 in other types of cells but not type 1 in skeletal muscle (38). The ability of cADPR to sensitize these receptors to the stimulatory effect of Ca2+ provides a possible mechanism for intracellular Ca2+ mobilization and thus for the generation of both calcium oscillation and calcium waves. Through video-imaging analysis of fura-2-loaded adrenal chromaffin cells, it has been shown that the [Ca2+]i increase brought about by Ca2+ influx through plasma membrane by nicotinic agonists or high K+ was initially restricted to a subplasmalemmal region. This restricted increase was then followed by more widespread elevation of [Ca2+]i throughout the cytoplasm (41). Thus, it was hypothesized in chromaffin cells that the second phase of the Ca2+ increase was due to the release of Ca2+ from internal stores by a CICR-dependent mechanism.

In this study, we demonstrated that 1) cADPR mediates the sensitization of CICR, 2) cADPR metabolism is under the control of cell activation, and 3) cADPR/CICR pathway forms a positive feedback loop in secretory response in adrenal chromaffin cells.

cADPR-induced Ca2+ Release

Our findings of the cross-potentiation of the effect of cADPR with caffeine or ryanodine but not with IP3 and of the specific antagonism of the effect of cADPR by a ryanodine receptor antagonist suggest that cADPR releases Ca2+ via a different mechanism from that of IP3. Chromaffin cells, therefore, seem to possess two functionally distinct Ca2+ stores sensitive to either IP3 or cADPR and caffeine. The different sensitivity to thapsigargin-induced inhibition of IP3- and of caffeine- or cADPR-induced Ca2+ release in chromaffin cells agrees well with the conclusion that the target pools for cADPR and IP3 are distributed differently within cells.

Phosphorylation of ryanodine receptors and of IP3 receptors by calmodulin and by A-PK have been shown in various tissues, but the regulation of Ca2+ release through phosphorylation was unclear. In the present study, the potency of cADPR but not of IP3 in releasing Ca2+ was enhanced by calmodulin in chromaffin cells. Both cADPR- and IP3-induced Ca2+ release were enhanced by A-PK. Lee et al. (42, 43) and Tanaka and Tashjian (44) suggest that cADPR requires the accessory or intermediate proteins to activate ryanodine receptors and actually identify two cADPR-binding proteins, 140 kDa and 100 kDa, in egg microsomes (45). Although it is not evident whether cADPR directly interacts with ryanodine receptor proteins or indirectly through its additional target factor(s) in adrenal chromaffin cells, Ca2+ release mediated by a cADPR-dependent process seems to receive positive modulations by A-PK and/or calmodulin in the cells.

Regulation of cADPR Synthesis

The membrane-bound protein that possesses ADP-ribosyl cyclase activities has been purified from lymphocyte, a 40-kDa protein called CD38, a lymphocyte surface antigen, and also from the spleen, a 39-kDa protein. Moreover, these proteins have cADPR hydrolase activities (28, 46). The amino acid sequence of the Aplysia ADP-ribosyl cyclase has been determined (47) and found to be homologous to CD38. Takasawa et al. (48) and Kato et al. (49) stress the importance of CD38 as a regulator of the level of cADPR in pancreatic beta  cells, where ATP generated during glucose metabolism in islets inhibits the cADPR hydrolase activity of CD38, thereby increasing the accumulation of cADPR. However, the physiological role of expressing an enzyme bifunctionally metabolizing cADPR on the surface of lymphocytes has not yet been elucidated. Although various tissues have been found to possess the activity of ADP-ribosyl cyclase and hydrolase, especially the brain (5, 29, 50), little is known about the control of ADP-ribosyl cyclase. To our knowledge, the present results are the first demonstration that ADP-ribosyl cyclase is activated in response to ACh, a physiological stimulation in adrenal chromaffin cells using NAD+ and NGD+ as substrate. ACh stimulated not only ADP-ribosyl cyclase activities but also hydrolysis of cADPR. Therefore, it is obvious that the increase in ADP-ribosyl cyclase activities induced by ACh is not due to an inhibition of cADPR hydrolysis. Recently, increased cADPR formation has been described in intestinal longitudinal muscle upon cholecystokinin administration, although the process leading to ADP-ribosyl cyclase activation was not investigated (16).

The activation of ADP-ribosyl cyclase by ACh appears to be Ca2+-dependent, because activation was blocked by omitting extracellular Ca2+ and also by an inhibitor of VOC. Therefore, it seems likely that the stimulation of ACh receptors does not directly activate the cyclase but regulates enzyme activity via the increase of [Ca2+]i. 8-Br-cAMP and forskolin mimic the ACh-induced activation of ADP-ribosyl cyclase. Moreover, blockers of A-PK blocked the activation of the cyclase activity induced by ACh, whereas forskolin-induced activation of the cyclase was not inhibited by the omission of external Ca2+ and the VOC inhibitor. We have previously demonstrated that ACh increases cAMP levels in the chromaffin cells by the activation of adenylate cyclase resulting from an increased influx of Ca2+ (39). Taken together, it was concluded that ACh elevates the Ca2+ influx through the plasma membrane in chromaffin cells, resulting in an activation of adenylate cyclase activity, and consequently, that the increased cAMP initiates the activation of ADP-ribosyl cyclase activity via a A-PK-dependent mechanism.

Activation of the cyclase induced by 8-Br-cAMP was characterized as an increase in initial velocity of the enzyme activity in intact cells, whereas a time-dependent progressive increase was observed in the assay in which 8-Br-cAMP was directly added in the homogenate containing the ATP generating system. Therefore, these findings suggest that the continuous phosphorylation of ADP-ribosyl cyclase or its related protein(s) by A-PK may be required for the maintenance of the cyclase activity. The deduced amino acid sequences of CD38 from human, mouse, and rat and of Aplysia ADP-ribosyl cyclase have several consensus phosphorylation sites for A-PK (38, 51).

Physiological Role of CICR in Stimulation-Secretion Coupling

cADPR-induced Ca2+ release in the nervous system has been demonstrated in brain microsomes (8-10). Its involvement in depolarization-induced increase in [Ca2+]i was also shown in bullfrog sympathetic ganglion cells (14) and in cultured Purkinje neurons (11), although the physiological significance has not been elucidated.

Our present findings suggest that the ADP-ribosyl cyclase/cADPR pathway contributes to the spread of Ca2+ to intracellular regions, which in turn regulate CA release response in chromaffin cells. This possibility was further supported by the specific inhibition by IpTxi of cADPR-induced Ca2+ release from permeabilized chromaffin cells and of the stimulation-evoked [Ca2+]i rise and CA release in intact cells. These effects of IpTxi seem unlikely to be due to the inhibition of Ca2+ influx through the plasma membrane because IpTxi did not affect the stimulation-evoked 45Ca2+ influx (data not shown).

ACh causes biphasic [Ca2+]i rise, an initial transient rise followed by sustained rise, in bovine adrenal chromaffin cells. Both phases are largely dependent on the presence of extracellular Ca2+ (52). Thus it is thought that Ca2+ influx through the plasma membrane is the main source for the [Ca2+]i rise caused by ACh in the cells. An analysis of the relationship between the peak height of [Ca2+]i and the time required for its half decay in the presence or absence of diltiazem and IpTxi revealed a clear difference in the mode of blockade of [Ca2+]i transient by these agents. Namely, diltiazem equally reduced the peak and sustained phases of ACh-induced [Ca2+]i rise, but IpTxi specifically reduced the sustained rise in [Ca2+]i. These results may reflect the differences in the mechanism of action between an inhibitor of VOC, diltiazem, and an inhibitor of CICR, IpTxi, suggesting that transient rise of [Ca2+]i is mainly due to a Ca2+ influx from the extracellular space and that the sustained rise of [Ca2+]i is constituted by a Ca2+ influx and a concurrently occurring Ca2+ release from intracellular stores by CICR.

Conclusions

It has been suggested that Ca2+ entry and the sustained rise of [Ca2+]i are essential for the activation of exocytosis in bovine chromaffin cells (52). Therefore, the present results suggest that the CICR pathway, which is activated through Ca2+ influx, contributes to the time-dependent rise of [Ca2+]i and to the maximal exocytotic response in chromaffin cells.

The evidence that cADPR is synthesized and degraded quickly in response to agonists, that cADPR stimulates the release of Ca2+ by a ryanodine-receptor-sensitive pathway, and that the physiological stimulation of [Ca2+]i transient and secretory response were blocked by an inhibitor of CICR satisfies criteria for cADPR as a second messenger of CICR in adrenal chromaffin cells.


FOOTNOTES

*   This work was supported in part by Grants-in-aid for Scientific Research C-07672017 from the Ministry of Education, Science, and Culture, Japan.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. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Tel.: 81-82-257-5640; Fax: 81-82-257-5644; E-mail: todohi{at}ipc.hiroshima-u.ac.jp.
1   The abbreviations used are: [Ca2+]i, intracellular free Ca2+ concentration; IP3, inositol 1,4,5-trisphosphate; CICR, Ca2+-induced Ca2+ release; NAD+, nicotinamide adenine dinucleotide; NGD+, nicotinamide guanine dinucleotide; cADPR, cADP-ribose; cGDPR, cGDP-ribose; ACh, acetylcholine; CA, catecholamine; DHB Bio-Rex 70, dihydroxyboronyl Bio-Rex 70; HPLC, high pressure liquid chromatography; 8-Br-cAMP, 8-bromo-cAMP; 8-Br-cGMP, 8-bromo-cGMP; A-PK, cAMP-dependent protein kinase; A-PK C.S., catalytic subunit of A-PK; IpTxi, imperatoxin inhibitor; VOC, voltage-operated Ca2+ channel; cAMP-S, adenosine cyclic 3':5'-phosphorothioate; Rp-cAMP-S, Rp-diastereomer of cAMP-S, sodium salt; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol.
2   K. Morita, S. Kitayama, and T. Dohi, unpublished observations.

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